Methods of determining the level of human tumor necrosis factor receptor-like 2

ABSTRACT

The present invention relates to novel members of the Tumor Necrosis Factor family of receptors. The invention provides isolated nucleic acid molecules encoding a human TR2 receptor and two splice variants thereof. TR2 polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TR2 receptor activity. Also provided are diagnostic methods for detecting disease states related to the aberrant expression of TR2 receptors. Further provided are therapeutic methods for treating disease states related to aberrant proliferation and differentiation of cells which express the TR2 receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/340,690, filed Jun. 29, 1999 (now U.S. Pat. No. 7,446,169), which isa divisional of U.S. application Ser. No. 08/741,095, filed Oct. 30,1996 (now U.S. Pat. No. 7,427,492), which is a continuation-in-part ofU.S. application Ser. No. 08/464,595 (now abandoned), U.S. applicationSer. No. 08/462,962 (now abandoned), and U.S. application Ser. No.08/462,315 (now abandoned), each of which was filed Jun. 5, 1995; U.S.application Ser. Nos. 08/464,595, 08/462,962 and 08/462,315 each arecontinuations-in part of PCT/US95/05058, filed Apr. 27, 1995. Thepresent application is also a divisional of U.S. application Ser. No.08/741,095, filed Oct. 30, 1996 (now U.S. Pat. No. 7,427,492), which isa continuation-in-part of U.S. application Ser. No. 08/464,595, U.S.application Ser. No. 08/462,962, and U.S. application Ser. No.08/462,315, each of which was filed Jun. 5, 1995; U.S. application Ser.Nos. 08/464,595, 08/462,962 and 08/462,315 each are continuations-inpart of PCT/US95/05058, filed Apr. 27, 1995. The disclosure of each ofthe above-identified priority applications is hereby incorporated byreference herein in its entirety.

STATEMENT UNDER 37 C.F.R. §1.77(b)(5)

This application refers to a “Sequence Listing” listed below, which isprovided as a text document. The document is entitled“PF173P4D2_SeqListing.txt” (42,187 bytes, created Jun. 24, 2008), and ishereby incorporated by reference in its entirety herein.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel members of the Tumor NecrosisFactor (TNF) receptor family. More specifically, isolated nucleic acidmolecules are provided encoding a human TNF receptor-related protein,referred to herein as the TR2 receptor of FIG. 1A-1B, havingconsiderable homology to murine CD40. Two different TR2 splice variants,referred to as TR2-SV1 and TR2-SV2, are also provided. TR2 polypeptidesare also provided with homology to human type 2 TNF receptor (TNF-RII).Further provided are vectors, host cells and recombinant methods forproducing the same. The invention also relates to both the inhibitionand enhancement of the activity of TR2 receptor polypeptides anddiagnostic methods for detecting TR2 receptor gene expression.

2. Related Art

Human tumor necrosis factors α (TNF-α) and (TNF-β or lymphotoxin) arerelated members of a broad class of polypeptide mediators, whichincludes the interferons, interleukins and growth factors, collectivelycalled cytokines (Beutler, B. and Cerami, A., Annu. Rev. Immunol.,7:625-655 (1989)).

Tumor necrosis factor (TNF-α and TNF-β) was originally discovered as aresult of its anti-tumor activity, however, now it is recognized as apleiotropic cytokine playing important roles in a host of biologicalprocesses and pathologies. To date, there are ten known members of theTNF-related cytokine family, TNF-α, TNF-β (lymphotoxin-α), LT-β, TRAILand ligands for the Fas receptor, CD30, CD27, CD40, OX40 and 4-1BBreceptors. These proteins have conserved C-terminal sequences andvariable N-terminal sequences which are often used as membrane anchors,with the exception of TNF-β. Both TNF-α and TNF-β function ashomotrimers when they bind to TNF receptors.

TNF is produced by a number of cell types, including monocytes,fibroblasts, T-cells, natural killer (NK) cells and predominately byactivated macrophages. TNF-α has been reported to have a role in therapid necrosis of tumors, immunostimulation, autoimmune disease, graftrejection, producing an anti-viral response, septic shock, cerebralmalaria, cytotoxicity, protection against deleterious effects ofionizing radiation produced during a course of chemotherapy, such asdenaturation of enzymes, lipid peroxidation and DNA damage (Nata et al.,J. Immunol. 136(7):2483 (1987)), growth regulation, vascular endotheliumeffects and metabolic effects. TNF-α also triggers endothelial cells tosecrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 topromote cell proliferation. In addition, TNF-α up-regulates various celladhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF-α and theFas ligand have also been shown to induce programmed cell death.

TNF-β has many activities, including induction of an antiviral state andtumor necrosis, activation of polymorphonuclear leukocytes, induction ofclass I major histocompatibility complex antigens on endothelial cells,induction of adhesion molecules on endothelium and growth hormonestimulation (Ruddle, N. and Homer, R., Prog. Allergy 40:162-182 (1988)).

Both TNF-α and TNF-β are involved in growth regulation and interact withhemopoietic cells at several stages of differentiation, inhibitingproliferation of various types of precursor cells, and inducingproliferation of immature myelomonocytic cells. Porter, A., Tibtech9:158-162 (1991).

Recent studies with “knockout” mice have shown that mice deficient inTNF-β production show abnormal development of the peripheral lymphoidorgans and morphological changes in spleen architecture (reviewed inAggarwal et al., Eur Cytokine Netw, 7(2):93-124 (1996)). With respect tothe lymphoid organs, the popliteal, inguinal, para-aortic, mesenteric,axillary and cervical lymph nodes failed to develop in TNF-β−/− mice. Inaddition, peripheral blood from TNF-β−/− mice contained a three foldreduction in white blood cells as compared to normal mice. Peripheralblood from TNF-β−/− mice, however, contained four fold more B cells ascompared to their normal counterparts. Further, TNF-, in contrast toTNF-α has been shown to induce proliferation of EBV-infected B cells.These results indicate that TNF-β is involved in lymphocyte development.

The first step in the induction of the various cellular responsesmediated by TNF-α or TNF-β is their binding to specific cell surface orsoluble receptors. Two distinct TNF receptors of approximately 55-KDa(TNF-RI) and 75-KDa (TNF-RII) have been identified (Hohman et al., J.Biol. Chem., 264:14927-14934 (1989)), and human and mouse cDNAscorresponding to both receptor types have been isolated andcharacterized (Loetscher et al., Cell, 61:351 (1990)). Both TNF-Rs sharethe typical structure of cell surface receptors including extracellular,transmembrane and intracellular regions.

These molecules exist not only in cell bound forms, but also in solubleforms, consisting of the cleaved extra-cellular domains of the intactreceptors (Nophar et al., EMBO Journal, 9 (10):3269-76 (1990)) andotherwise intact receptors wherein the transmembrane domain is lacking.The extracellular domains of TNF-RI and TNF-RII share 28% identity andare characterized by four repeated cysteine-rich motifs with significantintersubunit sequence homology. The majority of cell types and tissuesappear to express both TNF receptors and both receptors are active insignal transduction, however, they are able to mediate distinct cellularresponses. Further, TNF-RII was shown to exclusively mediate humanT-cell proliferation by TNF as shown in PCT WO 94/09137.

TNF-RI dependent responses include accumulation of C-FOS, IL-6, andmanganese superoxide dismutase mRNA, prostaglandin E2 synthesis, IL-2receptor and MHC class I and II cell surface antigen expression, growthinhibition, and cytotoxicity. TNF-RI also triggers second messengersystems such as phospholipase A₂, protein kinase C,phosphatidylcholine-specific phospholipase C and sphingomyelinase(Pfeffer, K. et al., Cell, 73:457-467 (1993)).

Several interferons and other agents have been shown to regulate theexpression of TNF receptors. Retinoic acid, for example, has been shownto induce the production of TNF receptors in some cells type while downregulating production in other cells. In addition, TNF-α has been showneffect the localization of both types of receptor. TNF-α inducesinternalization of TNF-RI and secretion of TNF-RII (reviewed in Aggarwalet al., supra). Thus, the production and localization of both TNF-Rs areregulated by a variety of agents.

Both the yeast two hybrid system and co-precipitation and purificationhave been used to identify ligands which associate with both types ofthe TNF-Rs (reviewed in Aggarwal et al., supra and Vandenabeele et al.,Trends in Cell Biol. 5:392-399 (1995)). Several proteins have beenidentified which interact with the cytoplasmic domain of a murine TNF-R.Two of these proteins appear to be related to the baculovirus inhibitorof apoptosis, suggesting a direct role for TNF-R in the regulation ofprogrammed cell death.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding a TR2 receptor and splice variantsthereof having the amino acid sequences shown in FIG. 1A-1B (SEQ IDNO:2), FIG. 4A-4C (SEQ ID NO:5) and FIG. 7A-7C (SEQ ID NO:8) or theamino acid sequence encoded by the cDNA clone encoding the TR2 receptorsdeposited as ATCC™ Deposit Numbers 97059, 97058 and 97057 on Feb. 13,1995. The present invention also relates to recombinant vectors, whichinclude the isolated nucleic acid molecules of the present invention,and to host cells containing the recombinant vectors, as well as tomethods of making such vectors and host cells and for using them forproduction of TR2 polypeptides or peptides by recombinant techniques.

The invention further provides isolated TR2 polypeptides having aminoacid sequences encoded by the polynucleotides described herein.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby TR2 receptors, which involves contacting cells which express TR2receptors with the candidate compound, assaying a cellular response, andcomparing the cellular response to a standard cellular response, thestandard being assayed when contact is made in absence of the candidatecompound; whereby, an increased cellular response over the standardindicates that the compound is an agonist and a decreased cellularresponse over the standard indicates that the compound is an antagonist.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason the binding of cellular ligands to TR2 receptors. In particular, themethod involves contacting TR2 receptors with a ligand polypeptide and acandidate compound and determining whether ligand binding to the TR2receptors is increased or decreased due to the presence of the candidatecompound.

The invention further provides a diagnostic method useful duringdiagnosis or prognosis of a disease states resulting from aberrant cellproliferation due to alterations in TR2 receptor expression.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of a TR2 receptoractivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of isolatedTR2 polypeptides of the invention or an agonist thereof.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of a TR2 receptoractivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of a TR2receptor antagonist.

The invention additionally provides soluble forms of the polypeptides ofthe present invention. Soluble peptides are defined by amino acidsequences wherein the sequence comprises the polypeptide sequenceslacking a transmembrane domain. Such soluble forms of the TR2 receptorsare useful as antagonists of the membrane bound forms of the receptors.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A-1B shows the nucleotide (SEQ ID NO: 1) and deduced amino acid(SEQ ID NO:2) sequences of a TR2 receptor. The protein has a predictedleader sequence of about 36 amino acid residues (underlined) (amino acidresidues −36 to −1 in SEQ ID NO:2) and a deduced molecular weight ofabout 30,417 kDa. It is further predicted that amino acid residues fromabout 37 to about 200 (amino acid residues 1 to 164 in SEQ ID NO:2)constitute the extracellular domain; from about 201 to about 225 (aminoacid residues 165 to 189 in SEQ ID NO:2) the transmembrane domain(underlined); and from about 226 to about 283 (amino acid residues 190to 247 in SEQ ID NO:2) the intracellular domain. Two potentialasparagine-linked glycosylation sites are located at amino acidpositions 110 and 173 (amino acid residues 74 to 137 in SEQ ID NO:2).

FIG. 2 shows the regions of similarity between the amino acid sequencesof the TR2 receptor protein of FIG. 1A-1B and a murine CD40 protein (SEQID NO:3) (percent similarity: 46.591; percent identity: 28.788).

FIG. 3 shows an analysis of the TR2 receptor amino acid sequence of FIG.1A-1B. Alpha, beta, turn and coil regions; hydrophilicity andhydrophobicity; amphipathic regions; flexible regions; antigenic indexand surface probability are shown. In the “Antigenic Index—Jameson-Wolf”graph, amino acid residues 39 to 70, 106 to 120, 142 to 189 and 276 to283 in FIG. 1A-1B (amino acid residues 3 to 34, 70 to 84, 106 to 153 and240 to 247 in SEQ ID NO:2) correspond to the shown highly antigenicregions of the TR2 receptor protein.

FIG. 4A-4C shows the nucleotide (SEQ ID NO:4) and deduced amino acid(SEQ ID NO:5) sequences of the TR2-SV1 receptor. The protein has apredicted leader sequence of about 36 amino acid residues (underlined)(amino acid residues −36 to −1 in SEQ ID NO:5) and a deduced molecularweight of about 19.5 kDa.

FIG. 5 shows the regions of similarity between the amino acid sequencesof the full-length TR2-SV1 receptor protein and a human type 2 TNFreceptor (SEQ ID NO:6) (percent similarity: 47.541; percent identity:24.590).

FIG. 6 shows an analysis of the TR2-SV1 receptor amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 39 to 70, 99 to 136 and 171 to 185 in FIG. 4A-4C(amino acid residues 3 to 34, 63 to 100 and 135 to 149 in SEQ ID NO:5)correspond to the shown highly antigenic regions of the TR2-SV1 receptorprotein.

FIG. 7A-7C shows the nucleotide (SEQ ID NO:7) and deduced amino acid(SEQ ID NO:8) sequences of the TR2-SV2 receptor. This protein lacks aputative leader sequence and has a deduced molecular weight of about 14kDa.

FIG. 8 shows the regions of similarity between the amino acid sequencesof the TR2-SV2 receptor protein and a human type 2 TNF receptor (SEQ IDNO:9) (percent similarity: 45.522; percent identity: 26.866).

FIG. 9 shows an analysis of the TR2-SV2 receptor amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues 56 to 68 and 93 to 136 in FIG. 7A-7C (SEQ ID NO:8)correspond to the shown highly antigenic regions of the TR2-SV2 receptorprotein.

FIG. 10 shows the regions of similarity between the amino acid sequencesof the TR2 receptor protein of FIG. 1A-1B and the TR2-SV1 receptorprotein of FIG. 4A-4C (percent similarity: 73.370; percent identity:9.783).

FIG. 11 shows the regions of similarity between the amino acid sequencesof the TR2 receptor protein of FIG. 1A-1B and the TR2-SV2 receptorprotein of FIG. 7A-7C (percent similarity: 70.588; percent identity:60.294).

FIG. 12 shows the regions of similarity between the amino acid sequencesof the TR2-SV1 and the TR2-SV2 receptor proteins (percent similarity:37.984; percent identity: 20.155).

FIG. 13A-13D shows the regions of similarity between the nucleotidesequences encoding the TR2 receptor protein of FIG. 1A-1B and theTR2-SV1 receptor protein of FIG. 4A-4C (percent similarity: 92.168;percent identity: 92.168).

FIG. 14A-14D shows the regions of similarity between the nucleotidesequences encoding the TR2 receptor protein of FIG. 1A-1B and theTR2-SV2 receptor protein of FIG. 7A-7C.

FIG. 15A-15F shows the regions of similarity between the nucleotidesequences encoding the TR2-SV1 and the TR2-SV2 receptor proteins(percent similarity: 53.479; percent identity: 53.479).

FIG. 16 shows an alignment of the amino acid sequence of the TR2receptor of FIG. 1A-1B (SEQ ID NO:2) with other TNFR family members. Theamino acid sequence of TR2 was aligned with those of TNFR-I (SEQ ID NO:10), TNFR-II (SEQ ID NO:11), CD40 (SEQ ID NO: 12) and 4-1BB (SEQ ID NO:13) on the basis of sequence homology and conserved cysteine residues.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising polynucleotides encoding a TR2 polypeptide (FIG. 1A-1B (SEQID NO:2)) and splice variants thereof, TR2-SV1 (FIG. 4A-4C (SEQ IDNO:5)) and TR2-SV2 (FIG. 7A-7C (SEQ ID NO:8)), the amino acid sequencesof which were determined by sequencing cloned cDNAs. The TR2 proteinshown in FIG. 1A-1B shares sequence homology with the murine CD40receptor (FIG. 2 (SEQ ID NO:3)). On Feb. 13, 1995 a deposit was made atthe American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110-2209, USA, and given accession number 97059. Thenucleotide sequence shown in FIG. 1A-1B (SEQ ID NO: 1) was obtained bysequencing a cDNA clone (Clone ID HLHAB49) containing the same aminoacid coding sequences as the clone in ATCC™ Accession No. 97059 withminor deviation. The cDNA sequence shown in FIG. 1A-1B (SEQ ID NO: 1)differs from that of the ATCC™ deposit in the 5′ and 3′ noncodingnucleotide sequences and three nucleotides.

The clone deposited in ATCC™ Accession No. 97059 contains 8 nucleotides5′ to the TR2 initiation codon and 21 nucleotides 3′ to the TR2 stopcodon. In contrast, the TR2 cDNA sequence of HLHAB49, shown in FIG.1A-1B (SEQ ID NO: 1), contains considerably longer non-codingnucleotides sequence on both the 5′ and 3′ ends of the TR2 codingsequences. Further, the TR2 receptor nucleotide sequence shown in FIG.1A-1B (SEQ ID NO: 1) contains an adenine at nucleotide 314, a cytosineat nucleotide 386, and a cytosine at nucleotide 627. In contrast, theclone of ATCC™ Accession No. 97059 contains a guanine at nucleotide 314,a thymine at nucleotide 386, and a thymine at nucleotide 627.

The TR2 receptors of the present invention include several allelicvariants containing alterations in at least these three nucleotides andtwo amino acids. Nucleotide sequence variants which have been identifiedinclude either guanine or adenine at nucleotide 314, thymine or cytosineat nucleotide 386, and thymine or cytosine at nucleotide 627 shown inFIG. 1A-1B (SEQ ID NO: 1). While the identified alteration at nucleotide627 is silent, the alteration at nucleotide 386 results in the codon atnucleotides 385 to 387 encoding either serine or phenylalanine and thealteration at nucleotide 314 results in the codon at nucleotides 313 to315 encoding either lysine or arginine.

The nucleotide sequences shown in FIG. 4A-4C (SEQ ID NO:4) and FIG.7A-7C (SEQ ID NO:7) were also obtained by sequencing cDNA clonesdeposited on Feb. 13, 1995 at the American Type Culture Collection andgiven accession numbers 97058 (TR2-SV1) and 97057 (TR2-SV2),respectively. All of the deposited clones are contained in thepBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.).

As used herein the phrase “splice variant” refers to cDNA moleculesproduced from a RNA molecules initially transcribed from the samegenomic DNA sequence which have undergone alternative RNA splicing.Alternative RNA splicing occurs when a primary RNA transcript undergoessplicing, generally for the removal of introns, which results in theproduction of more than one mRNA molecule each of which may encodedifferent amino acid sequences. The term “splice variant” also refers tothe proteins encoded by the above cDNA molecules.

As used herein, “TR2 proteins”, “TR2 receptors”, “TR2 receptor proteins”and “TR2 polypeptides” refer to all proteins resulting from thealternate splicing of the genomic DNA sequences encoding proteins havingregions of amino acid sequence identity and receptor activity whichcorrespond to the proteins shown in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C(SEQ ID NO:5) or FIG. 7A-7C (SEQ ID NO:8). The TR2 protein shown in FIG.1A-1B, the TR2-SV1 protein shown FIG. 4A-4C and the TR2-SV2 proteinshown in FIG. 7A-7C are examples of such receptor proteins.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequencein FIG. 1A-1B, FIG. 4A-4C or FIG. 7A-7C, nucleic acid molecules of thepresent invention encoding TR2 polypeptides may be obtained usingstandard cloning and screening procedures, such as those used forcloning cDNAs using mRNA as starting material. Illustrative of theinvention, the nucleic acid molecule described in FIG. 1A-1B (SEQ IDNO: 1) was discovered in a cDNA library derived from activated humanT-lymphocytes. The nucleic acid molecules described in FIG. 4A-4C (SEQID NO:4) and FIG. 7A-7C (SEQ ID NO:7) were discovered in cDNAs libraryderived from human fetal heart and human stimulated monocytes,respectively.

As described in Example 6, TR2 mRNA was detected in numerous tissuesincluding lung, spleen and thymus and may be ubiquitously expressed inhuman cells. TR2RNA was also found to be expressed in B lymphocytes(CD19⁺), both CD4⁺ (T_(H1) and T_(H2) clones) and CD8⁺ T lymphocytes,monocytes and endothelial cells.

As also noted in Example 6, the production of TR2 mRNA was inducible inMG 63 cells by TNF-α. Further, the accumulation of TR2 mRNA was observedin HL60, U937 and THP1 cells upon PMA or DMSO treatment. PMA and DMSOare agents known to induce differentiation of these three cell types.

The determined nucleotide sequence of the TR2 cDNA of FIG. 1A-1B (SEQ IDNO: 1) contains an open reading frame encoding a protein of about 283amino acid residues, with a predicted leader sequence of about 36 aminoacid residues, and a deduced molecular weight of about 30,417 kDa. Theamino acid sequence of the predicted mature TR2 receptor is shown inFIG. 1A-1B from amino acid residue about 37 to residue about 283 (aminoacid residues 1 to 247 in SEQ ID NO:2). As noted in Example 6, thelocation of the leader sequence cleavage site was confirmed for a TR2-Fcfusion protein and found to be between amino acids 36 and 37 shown inFIG. 1A-1B (amino acid residues −1 to 1 in SEQ ID NO:2). The TR2 proteinshown in FIG. 1A-1B (SEQ ID NO:2) is about 29% identical and about 47%similar to the murine CD40 protein shown in SEQ ID NO:3 (see FIG. 2).

Similarly, the determined cDNA nucleotide sequences of the TR2-SV1splice variant of TR2 (FIG. 4A-4C (SEQ ID NO:4)) contains an openreading frame encoding a protein of about 185 amino acid residues, witha predicted leader sequence of about 36 amino acid residues, and adeduced molecular weight of about 19.5 kDa. The amino acid sequence ofthe predicted mature TR2-SV1 receptor is shown in FIG. 4A-4C (SEQ IDNO:5) from amino acid residue about 37 to residue about 185 (amino acidresidues 1 to 149 in (SEQ ID NO:5). The TR2-SV1 protein shown in FIG.4A-4C (SEQ ID NO:5) is about 25% identical and about 48% similar to thehuman type 2 TNF receptor protein shown in SEQ ID NO:6 (see FIG. 5).

The determined cDNA nucleotide sequences of the TR2-SV2 splice variantof TR2 (FIG. 7A-7C (SEQ ID NO:7)) contains an open reading frameencoding a protein of about 136 amino acid residues, without a predictedleader sequence, and a deduced molecular weight of about 14 kDa. Theamino acid sequence of the predicted TR2-SV2 receptor is shown in FIG.7A-7C (SEQ ID NO:8) from amino acid residue about 1 to residue about136. The TR2-SV2 protein shown in FIG. 7A-7C (SEQ ID NO:8) is about 27%identical and about 45% similar to the human type 2 TNF receptor proteinshown in SEQ ID NO: 9 (see FIG. 8).

A comparison of both the nucleotide and amino acid sequences of the TR2,TR2-SV1 and TR2-SV2 receptor proteins shown in FIG. 1A-1B, FIG. 4A-4Cand FIG. 7A-7C shows several regions of near identity. While the aminoacid sequence of the TR2 receptor protein, shown in FIG. 1A-1B (SEQ IDNO:2), is about 60% identical and about 73% similar to the amino acidsequence of the TR2-SV1 receptor protein, shown in FIG. 4A-4C (SEQ IDNO:5), in approximately the first one hundred amino acids of theirrespective sequences the two proteins differ in one location (FIG. 10).

Similarly, the amino acid sequence of the TR2 receptor protein of FIG.1A-1B (SEQ ID NO:2) is about 60% identical and about 71% similar to theamino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C(SEQ ID NO:8); however, the two proteins are almost identical over a 60amino acid stretch in the central portion of the TR2-SV2 protein (FIG.11).

In contrast, the TR2-SV1 and TR2-SV2 proteins are only about 20%identical and about 38% similar at the amino acid level to each other.Unlike the comparisons of either of these proteins to the TR2 proteinshown in FIG. 1A-1B (SEQ ID NO:2), these proteins share their homologyover the entire 136 amino acid sequence of the TR2-SV2 protein (FIG.12).

With respect to their nucleotide sequences of the cDNAs encoding thedisclosed TR2 proteins, a comparison of these sequences indicates thatthe TR2 cDNAs share large regions of near identity at the nucleic acidlevel (FIG. 13A-13D, FIG. 14A-13C and FIG. 15A-15F). The cDNA sequencesencoding the TR2 and TR2-SV1 proteins, for example, share large regionsof near identity in their nucleotide sequences which encode both the Ntermini of the respective proteins and their 5′ and 3′ noncoding regions(FIG. 13A-13D). Further, the nucleotide sequences of the cDNAs encodingthe TR2-SV1 and TR2-SV2 proteins share considerable homology but thisidentity is limited to their 3′ regions well beyond their respectivecoding sequences (FIG. 15A-15F).

Such regions of near identity between two different cDNA sequences, whenmaintained over an extended stretch of sequence, indicates to oneskilled in the art that the respective molecules were originallytranscribed from the same genomic DNA sequence. One skilled in the artwould further recognize that, since more than one codon can encode thesame amino acid, identity between two proteins at the amino acid leveldoes not necessarily mean that the DNA sequences encoding those proteinswill share similar regions of identity. The above data indicates thatthe TR2 receptors of the present invention are transcribed from a singlegenomic DNA sequence and represent multiple splice variants of oneinitial RNA transcript.

Related proteins which are produced from alternately spliced RNA,referred to as splice variants, are known in the art. The transcript ofthe src gene, for example, undergoes alternate RNA splicing to producecell type specific products. In most cells the Src protein consists of533 amino acids while in nerve cells an additional short exon isincluded in the mRNA resulting in a protein of 539 amino acids. SeeAlberts, B. et al., MOLECULAR BIOLOGY OF THE CELL (3rd Edition, GarlandPublishing, Inc., 1994), 455. Similarly, sex specific mRNA transcriptshave been identified in Drosophila where alternate mRNA splicing resultsin a protein named Dsx which is approximately 550 amino acids in lengthin males and 430 amino acids in length in females. These two splicevariant proteins share a common core sequence of about 400 amino acids.See id. at 457.

In the present instance, the TR2 receptor protein shown in FIG. 1A-1B(SEQ ID NO:2) is believed to be the full-length polypeptide encoded bythe RNA from which the TR2 receptor proteins are translated. The RNAencoding the TR2-SV1 splice variant shown in FIG. 4A-4C (SEQ ID NO:5) isbelieved to contain an insertion in the region encoding amino acidresidue 102 of the amino acid sequence shown in FIG. 1A-1B and adeletion in the region encoding amino acid residue 184 of the amino acidsequence shown in FIG. 1A-1B. The RNA encoding the TR2-SV2 splicevariant shown in FIG. 7A-7C is believed to begin with the nucleotidesequence encoding amino acid residue 102 of the amino acid sequenceshown in FIG. 1A-1B and contain insertions in the regions encoding aminoacid residues 184 and 243 of the amino acid sequence shown in FIG.1A-1B.

As indicated, the present invention also provides the mature forms ofthe TR2 receptors of the present invention. According to the signalhypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.Therefore, the present invention provides nucleotide sequences encodingmature TR2 polypeptides having the amino acid sequences encoded by thecDNA clones contained in the host identified as ATCC™ Deposit Numbers97059 and 97058 and as shown in FIG. 1A-1B (SEQ ID NO:2) and FIG. 4A-4C(SEQ ID NO:5). By the mature TR2 polypeptides having the amino acidsequences encoded by the cDNA clones contained in the host identified asATCC™ Deposit Numbers 97059 and 97058 is meant the mature form(s) of theTR2 receptors produced by expression in a mammalian cell (e.g., COScells, as described below) of the complete open reading frame encoded bythe human DNA sequence of the clone contained in the vector in thedeposited host.

The invention also provides nucleic acid sequences encoding the TR2-SV2receptor protein of FIG. 7A-7C (SEQ ID NO:8), having the amino acidsequence encoded by the cDNA clone contained in ATCC™ Deposit Number97057, which does not contain a secretory leader sequence.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequences of the completeTR2 polypeptides shown in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ IDNO:5) and FIG. 7A-7C (SEQ ID NO:8) were analyzed by a computer program(“PSORT”) (K. Nakai and M. Kanehisa, Genomics 14:897-911 (1992)), whichis an expert system for predicting the cellular location of a proteinbased on the amino acid sequence. As part of this computationalprediction of localization, the methods of McGeoch and von Heinje areincorporated. The analysis by the PSORT program predicted the cleavagesites between amino acids −1 and 1 in SEQ ID NO:2 and SEQ ID NO:5.Thereafter, the complete amino acid sequences were further analyzed byvisual inspection, applying a simple form of the (−1, −3) rule of vonHeine. von Heinje, supra. Thus, the leader sequences for the TR2 proteinshown in SEQ ID NO:2 and the TR2-SV1 protein are predicted to consist ofamino acid residues −36 to −1 in both SEQ ID NO:2 and SEQ ID NO:5, whilethe predicted mature TR2 proteins consist of amino acid residues 1 to247 for the TR2 protein shown in SEQ ID NO:2 and residues 1 to 149 forthe TR2-SV1 protein shown in SEQ ID NO:5.

As noted in Example 6, the cleavage site of the leader sequence of aTR2-Fc fusion protein was confirmed using amino acid analysis of theexpressed fusion protein. This fusion protein was found to begin atamino acid 37, which corresponds to amino acid 1 in SEQ ID NO:2 and SEQID NO:5, indicating that the cleavage site of the leader sequence isbetween amino acids 36 and 37 in this protein (corresponding to aminoacid residues −1 to 1 in SEQ ID NO:2 and SEQ ID NO:5).

As one of ordinary skill would appreciate, however, due to thepossibilities of sequencing errors, as well as the variability ofcleavage sites for leaders in different known proteins, the TR2 receptorpolypeptide encoded by the cDNA of ATCC™ Deposit Number 97059 comprisesabout 283 amino acids, but may be anywhere in the range of 250 to 316amino acids; and the leader sequence of this protein is about 36 aminoacids, but may be anywhere in the range of about 30 to about 42 aminoacids. Similarly, the TR2-SV1 receptor polypeptide encoded by the cDNAof ATCC™ Deposit Number 97058 comprises about 185 amino acids, but maybe anywhere in the range of 163-207 amino acids; and the leader sequenceof this protein is about 36 amino acids, but may be anywhere in therange of about 30 to about 42 amino acids. Further, the TR2-SV2 receptorpolypeptide encoded by the cDNA of ATCC™ Deposit Number 97057 comprisesabout 136 amino acids, but may be anywhere in the range of 120-152 aminoacids.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its nativeenvironment. For example, recombinant DNA molecules contained in avector are considered isolated for the purposes of the presentinvention. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe DNA molecules of the present invention. Isolated nucleic acidmolecules according to the present invention further include suchmolecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) shown in FIG. 1A-1B(SEQ ID NO:1); DNA molecules comprising the coding sequence for themature TR2 receptor shown in FIG. 1A-1B (SEQ ID NO:2) (last 247 aminoacids); and DNA molecules which comprise a sequence substantiallydifferent from those described above but which, due to the degeneracy ofthe genetic code, still encode the TR2 receptor protein shown in FIG.1A-1B (SEQ ID NO:2). Of course, the genetic code is well known in theart. Thus, it would be routine for one skilled in the art to generatesuch degenerate variants.

Similarly, isolated nucleic acid molecules of the present inventioninclude DNA molecules comprising an open reading frame (ORF) shown inFIG. 4A-4C (SEQ ID NO:4); DNA molecules comprising the coding sequencefor the mature TR2-SV1 receptor shown in FIG. 4A-4C (SEQ ID NO:5) (last149 amino acids); and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode the TR2-SV1 receptor.

Further, isolated nucleic acid molecules of the present inventioninclude DNA molecules comprising an open reading frame (ORF) shown inFIG. 7A-7C (SEQ ID NO:7) and DNA molecules which comprise a sequencesubstantially different from those described above but which, due to thedegeneracy of the genetic code, still encode the TR2-SV2 receptor.

In another aspect, the invention provides isolated nucleic acidmolecules encoding the TR2, TR2-SV1 and TR2-SV2 polypeptides having theamino acid sequences encoded by the cDNA clones contained in the plasmiddeposited as ATCC™ Deposit No. 97059, 97058 and 97057, respectively, onFeb. 13, 1995. In a further embodiment, these nucleic acid moleculeswill encode a mature polypeptide or the full-length polypeptide lackingthe N-terminal methionine. The invention further provides isolatednucleic acid molecules having the nucleotide sequences shown in FIG.1A-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4), and FIG. 7A-7C (SEQ IDNO:7); the nucleotide sequences of the cDNAs contained in theabove-described deposited clones; or nucleic acid molecules having asequence complementary to one of the above sequences. Such isolatedmolecules, particularly DNA molecules, are useful as probes for genemapping, by in situ hybridization with chromosomes, and for detectingexpression of the TR2 receptor genes of the present invention in humantissue, for instance, by Northern blot analysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNAs or the nucleotide sequence shown in FIG. 1A-1B (SEQ ID NO: 1),FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NO:7) is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-400 nt in length are also useful according to the presentinvention as are fragments corresponding to most, if not all, of thenucleotide sequences of the deposited cDNAs or as shown in FIG. 1A-1B(SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NO:7).By a fragment at least 20 nt in length, for example, is intendedfragments which include 20 or more contiguous bases from the nucleotidesequences of the deposited cDNAs or the nucleotide sequences as shown inFIG. 1A-1B (SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQID NO:7).

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the TR2receptor protein of FIG. 1A-1B (SEQ ID NO:2) extracellular domain(predicted to constitute amino acid residues from about 37 to about 200in FIG. 1A-1B (amino acid residues 1 to 164 in SEQ ID NO:2)); apolypeptide comprising the TR2 receptor transmembrane domain (amino acidresidues 201 to 225 in FIG. 1A-1B (amino acid residues 165 to 189 in SEQID NO:2)); a polypeptide comprising the TR2 receptor intracellulardomain (predicted to constitute amino acid residues from about 226 toabout 283 in FIG. 1A-1B (amino acid residues 190 to 247 in SEQ IDNO:2)); and a polypeptide comprising the TR2 receptor protein of FIG.1A-1B (SEQ ID NO:2) extracellular and intracellular domains with all orpart of the transmembrane domain deleted.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding amino acid residues 1 to 162 of SEQ IDNO:26 and amino acid residues −38 to 162 of SEQ ID NO:26.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding polypeptides comprising the matureTR2-SV1 receptor (predicted to constitute amino acid residues from about37 to about 185 in FIG. 4A-4C (amino acid residues 1 to 149 in SEQ IDNO:5)) and the complete TR2-SV2 receptor (predicted to constitute aminoacid residues from about 1 to about 136 in FIG. 7A-7C (SEQ ID NO:8)).

As above with the leader sequence, the amino acid residues constitutingthe extracellular, transmembrane and intracellular domains have beenpredicted by computer analysis. Thus, as one of ordinary skill wouldappreciate, the amino acid residues constituting these domains may varyslightly (e.g., by about 1 to about 15 amino acid residues) depending onthe criteria used to define each domain.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding epitope-bearing portions of the TR2receptor proteins. In particular, such nucleic acid fragments of thepresent invention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 39 to about 70 in FIG. 1A-1B(amino acid residues 3 to 34 in SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 106 to about 120 in FIG. 1 (amino acidresidues 70 to 84 in SEQ ID NO:2); a polypeptide comprising amino acidresidues from about 142 to about 189 in FIG. 1A-1B (amino acid residues106 to 153 in SEQ ID NO:2); a polypeptide comprising amino acid residuesfrom about 276 to about 283 in FIG. 1A-1B (amino acid residues 240 to247 in SEQ ID NO:2); a polypeptide comprising amino acid residues fromabout 39 to about 70 in FIG. 4A-4C (amino acid residues 3 to 34 in SEQID NO:5); amino acid residues from about 99 to about 136 in FIG. 4A-4C(amino acid residues 63 to 100 in SEQ ID NO:5); amino acid residues fromabout 171 to about 185 in FIG. 4A-4C (amino acid residues 135 to 149 inSEQ ID NO:5); amino acid residues from about 56 to about 68 in FIG.7A-7C (SEQ ID NO:8); amino acid residues from about 93 to about 136 inFIG. 7A-7C (SEQ ID NO:8). The inventors have determined that the abovepolypeptide fragments are antigenic regions of the TR2 receptors.Methods for determining other such epitope-bearing portions of the TR2proteins are described in detail below.

In another aspect, the invention provides isolated nucleic acidmolecules comprising polynucleotides which hybridizes under stringenthybridization conditions to a portion of the polynucleotide of one ofthe nucleic acid molecules of the invention described above, forinstance, the cDNA clones contained in ATCC™ Deposits 97059, 97058 and97057. By “stringent hybridization conditions” is intended overnightincubation at 42° C. in a solution comprising: 50% formamide, 5×SSC (750mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNAs or the nucleotide sequences as shown in FIG. 1A-1B (SEQ ID NO: 1),FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NO:7)).

Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3 terminal poly(A) tract of the TR2 receptor cDNA sequencesshown in FIG. 1A-1B (SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or FIG.7A-7C (SEQ ID NO:7)), or to a complementary stretch of T (or U) resides,would not be included in a polynucleotide of the invention used tohybridize to a portion of a nucleic acid of the invention, since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

As indicated, nucleic acid molecules of the present invention whichencode TR2 polypeptides may include, but are not limited to thoseencoding the amino acid sequences of the mature polypeptides, by itself,the coding sequence for the mature polypeptides and additionalsequences, such as those encoding the about 36 amino acid leader orsecretory sequences, such as pre-, or pro- or prepro-protein sequences;the coding sequence of the mature polypeptides, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5′ and 3′ sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptides may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37: 767 (1984).As discussed below, other such fusion proteins include the TR2 receptorsfused to IgG-Fc at the N- or C-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the TR2 receptors. Variants may occur naturally, such asa natural allelic variant. By an “allelic variant” is intended one ofseveral alternate forms of a gene occupying a given locus on achromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons,New York (1985). Non-naturally occurring variants may be produced usingart-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of the TR2receptors or portions thereof. Also especially preferred in this regardare conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the TR2 polypeptidehaving the complete amino acid sequence shown in FIG. 1A-1B (amino acidresidues −36 to 247 in SEQ ID NO:2), FIG. 4A-4C (amino acid residues −36to 149 in SEQ ID NO:5), or FIG. 7A-7C (amino acid residues 1 to 136 inSEQ ID NO:8); (b) a nucleotide encoding the complete amino sequenceshown in FIG. 1A-1B (amino acid residues −35 to 247 in SEQ ID NO:2),FIG. 4A-4C (amino acid residues −35 to 149 in SEQ ID NO:5), or FIG.7A-7C (amino acid residues 2 to 136 in SEQ ID NO:8) but lacking theN-terminal methionine; (c) a nucleotide sequence encoding the mature TR2receptors (full-length polypeptide with any attending leader sequenceremoved) having the amino acid sequence at positions from about 37 toabout 283 in FIG. 1A-1B (amino acid residues 1 to 247 in SEQ ID NO:2) orthe amino acid sequence at positions from about 37 to about 185 in FIG.4A-4C (amino acid residues 1 to 149 in SEQ ID NO:5), or the amino acidsequence at positions from about 1 to about 136 in FIG. 7A-7C (SEQ IDNO:8); (d) a nucleotide sequence encoding the TR2, TR2-SV1 or TR2-SV2polypeptides having the complete amino acid sequence including theleader encoded by the cDNA clones contained in ATCC™ Deposit Numbers97059, 97058, and 97057, respectively; (e) a nucleotide sequenceencoding the mature TR2 or TR2-SV1 receptors having the amino acidsequences encoded by the cDNA clones contained in ATCC™ Deposit Numbers97059 and 97058, respectively; (f) a nucleotide sequence encoding theTR2 or TR2-SV1 receptor extracellular domain; (g) a nucleotide sequenceencoding the TR2 receptor transmembrane domain; (h) a nucleotidesequence encoding the TR2 receptor intracellular domain; (i) anucleotide sequence encoding the TR2 receptor extracellular andintracellular domains with all or part of the transmembrane domaindeleted; and (j) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TR2polypeptide is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the TR2receptors. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. These mutations of thereference sequence may occur at the 5 or 3 terminal positions of thereference nucleotide sequence or anywhere between those terminalpositions, interspersed either individually among nucleotides in thereference sequence or in one or more contiguous groups within thereference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in FIG. 1A-1B (SEQ ID NO: 1) or to thenucleotides sequence of the deposited cDNA clone encoding that proteincan be determined conventionally using known computer programs such asthe Bestfit program (Wisconsin Sequence Analysis Package, Version 8 forUnix, Genetics Computer Group, University Research Park, 575 ScienceDrive, Madison, Wis. 53711. Bestfit uses the local homology algorithm ofSmith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981),to find the best segment of homology between two sequences. When usingBestfit or any other sequence alignment program to determine whether aparticular sequence is, for instance, 95% identical to a referencesequence according to the present invention, the parameters are set, ofcourse, such that the percentage of identity is calculated over the fulllength of the reference nucleotide sequence and that gaps in homology ofup to 5% of the total number of nucleotides in the reference sequenceare allowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequencesshown in FIG. 1A-1B (SEQ ID NO:1), FIG. 4A-4C (SEQ ID NO:4), or FIG.7A-7C (SEQ ID NO:7) or to the nucleic acid sequence of the depositedcDNAs, irrespective of whether they encode a polypeptide having TR2receptor activity. This is because even where a particular nucleic acidmolecule does not encode a polypeptide having TR2 receptor activity, oneof skill in the art would still know how to use the nucleic acidmolecule, for instance, as a hybridization probe or a polymerase chainreaction (PCR) primer. Uses of the nucleic acid molecules of the presentinvention that do not encode a polypeptide having TR2 receptor activityinclude, inter alia, (1) isolating a TR2 receptor gene or allelic orsplice variants thereof in a cDNA library; (2) in situ hybridization(e.g., “FISH”) to metaphase chromosomal spreads to provide precisechromosomal location of a TR2 receptor gene, as described in Verma etal., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press,New York (1988); and (3) Northern Blot analysis for detecting TR2receptor mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in FIG. 1A-1B (SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or FIG.7A-7C (SEQ ID NO:7) or to the nucleic acid sequence of the depositedcDNAs which do, in fact, encode a polypeptide having TR2 receptoractivity. By “a polypeptide having TR2 receptor activity” is intendedpolypeptides exhibiting activity similar, but not necessarily identical,to an activity of the TR2 receptors of the present invention (either thefull-length protein, the splice variants, or, preferably, the matureprotein), as measured in a particular biological assay. For example, TR2receptor activity can be measured by determining the ability of apolypeptide-Fc fusion protein to inhibit lymphocyte proliferation asdescribed below in Example 6. TR2 receptor activity may also be measuredby determining the ability of a polypeptide, such as cognate ligandwhich is free or expressed on a cell surface, to confer proliferatoryactivity in intact cells expressing the receptor.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to the nucleic acid sequence of the depositedcDNAs or the nucleic acid sequences shown in FIG. 1A-1B (SEQ ID NO: 1),FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NO:7) will encodepolypeptides “having TR2 receptor activity.” In fact, since degeneratevariants of any of these nucleotide sequences all encode the samepolypeptide, this will be clear to the skilled artisan even withoutperforming the above described comparison assay. It will be furtherrecognized in the art that, for such nucleic acid molecules that are notdegenerate variants, a reasonable number will also encode a polypeptidehaving TR2 protein activity. This is because the skilled artisan isfully aware of amino acid substitutions that are either less likely ornot likely to significantly affect protein function (e.g., replacing onealiphatic amino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of TR2polypeptides or fragments thereof by recombinant techniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate heterologous hosts include, butare not limited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as antigen forimmunizations. In drug discovery, for example, human proteins, such as,human hIL-5 receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 (1995)and K. Johanson et al., The Journal of Biological Chemistry, Vol. 270,No. 16:9459-9471 (1995).

TR2 receptors can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification. Polypeptides of the presentinvention include naturally purified products, products of chemicalsynthetic procedures, and products produced by recombinant techniquesfrom a prokaryotic or eukaryotic host, including, for example,bacterial, yeast, higher plant, insect and mammalian cells. Dependingupon the host employed in a recombinant production procedure, thepolypeptides of the present invention may be glycosylated or may benon-glycosylated. In addition, polypeptides of the invention may alsoinclude an initial modified methionine residue, in some cases as aresult of host-mediated processes.

TR2Polypeptides and Fragments

The invention further provides isolated TR2 polypeptides having theamino acid sequence encoded by the deposited cDNAs, or the amino acidsequence in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), or FIG.7A-7C (SEQ ID NO:8), or a peptide or polypeptide comprising a portion ofthe above polypeptides.

The polypeptides of this invention may be membrane bound or may be in asoluble circulating form. Soluble peptides are defined by amino acidsequence wherein the sequence comprises the polypeptide sequence lackingthe transmembrane domain. One example of such a soluble form of the TR2receptor is the TR2-SV1 splice variant which has a secretory leadersequence but lacks both the intracellular and transmembrane domains.Thus, the TR2-SV1 receptor protein appears to be secreted in a solubleform from cells which express this protein.

The polypeptides of the present invention may exist as a membrane boundreceptor having a transmembrane region and an intra- and extracellularregion or they may exist in soluble form wherein the transmembranedomain is lacking. One example of such a form of the TR2 receptor is theTR2 receptor shown in FIG. 1A-1B (SEQ ID NO:2) which contains, inaddition to a leader sequence, transmembrane, intracellular andextracellular domains. Thus, this form of the TR2 receptor appears to belocalized in the cytoplasmic membrane of cells which express thisprotein

It will be recognized in the art that some amino acid sequences of theTR2 receptors can be varied without significant effect to the structureor function of the protein. If such differences in sequence arecontemplated, it should be remembered that there will be critical areason the protein which determine activity. Thus, the invention furtherincludes variations of the TR2 receptors which show substantial TR2receptor activity or which include regions of TR2 proteins such as theprotein portions discussed below. Such mutants include deletions,insertions, inversions, repeats, and type substitutions. As indicatedabove, guidance concerning which amino acid changes are likely to bephenotypically silent can be found in Bowie, J. U., et al., “Decipheringthe Message in Protein Sequences: Tolerance to Amino AcidSubstitutions,” Science 247:1306-1310 (1990).

Thus, the fragment, derivative or analog of the polypeptides of FIG.1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), and FIG. 7A-7C (SEQ IDNO:8), or that encoded by the deposited cDNAs, may be (i) one in whichone or more of the amino acid residues are substituted with a conservedor non-conserved amino acid residue (preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substituent group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TR2 proteins. The prevention ofaggregation is highly desirable. Aggregation of proteins not onlyresults in a loss of activity but can also be problematic when preparingpharmaceutical formulations, because they can be immunogenic. (Pinckardet al., Clin Exp. Immunol. 2:331-340 (1967); Robbins et al., Diabetes36:838-845 (1987); Cleland et al. Crit. Rev. Therapeutic Drug CarrierSystems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, the TR2receptors of the present invention may include one or more amino acidsubstitutions, deletions or additions, either from natural mutations orhuman manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 CONSERVATIVE AMINO ACID SUBSTITUTIONS. Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the TR2 proteins of the present invention that areessential for function can be identified by methods known in the art,such as site-directed mutagenesis or alanine-scanning mutagenesis(Cunningham and Wells, Science 244:1081-1085 (1989)). The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as receptor binding or in vitro, or in vitro proliferativeactivity. Sites that are critical for ligand-receptor binding can alsobe determined by structural analysis such as crystallization, nuclearmagnetic resonance or photoaffinity labeling (Smith et al., J. Mol.Biol. 224:899-904 (1992) and de Vos et al. Science 255:306-312 (1992)).

The polypeptides of the present invention are preferably provided in anisolated form. By “isolated polypeptide”, is intended a polypeptideremoved from its native environment. Thus, a polypeptide produced andcontained within a recombinant host cell would be considered “isolated”for purposes of the present invention. Also intended as an “isolatedpolypeptide” are polypeptides that have been purified, partially orsubstantially, from a recombinant host. For example, recombinantlyproduced versions of the TR2 receptors can be substantially purified bythe one-step method described in Smith and Johnson, Gene 67:31-40(1988).

The polypeptides of the present invention also include the polypeptideencoded by the deposited cDNAs including the leader; the polypeptideencoded by the deposited the cDNAs minus the leader (i.e., the matureprotein); the polypeptides of FIG. 1A-1B (SEQ ID NO:2) or FIG. 4A-4C(SEQ ID NO:5) including the leader; the polypeptides of FIG. 1A-1B (SEQID NO:2) or FIG. 4A-4C (SEQ ID NO:5) including the leader but minus theN-terminal methionine; the polypeptides of FIG. 1A-1B (SEQ ID NO:2) orFIG. 4A-4C (SEQ ID NO:5) minus the leader; the polypeptide of FIG. 7A-7C(SEQ ID NO:8); the extracellular domain, the transmembrane domain, andthe intracellular domain of the TR2 receptor shown in FIG. 1A-1B (SEQ IDNO:2); and polypeptides which are at least 80% identical, morepreferably at least 90% or 95% identical, still more preferably at least96%, 97%, 98% or 99% identical to the polypeptides described above, andalso include portions of such polypeptides with at least 30 amino acidsand more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TR2 polypeptideis intended that the amino acid sequence of the polypeptide is identicalto the reference sequence except that the polypeptide sequence mayinclude up to five amino acid alterations per each 100 amino acids ofthe reference amino acid of a TR2 receptor. In other words, to obtain apolypeptide having an amino acid sequence at least 95% identical to areference amino acid sequence, up to 5% of the amino acid residues inthe reference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ IDNO:5), or FIG. 7A-7C (SEQ ID NO:8) or to the amino acid sequence encodedby one of the deposited cDNA clones can be determined conventionallyusing known computer programs such the Bestfit program (WisconsinSequence Analysis Package, Version 8 for Unix, Genetics Computer Group,University Research Park, 575 Science Drive, Madison, Wis. 53711). Whenusing Bestfit or any other sequence alignment program to determinewhether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parametersare set, of course, such that the percentage of identity is calculatedover the full length of the reference amino acid sequence and that gapsin homology of up to 5% of the total number of amino acid residues inthe reference sequence are allowed.

The polypeptides of the present invention could be used as a molecularweight marker on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art.

In another aspect, the invention provides peptides or polypeptidescomprising epitope-bearing portions of the polypeptides of theinvention. The epitopes of these polypeptide portions are an immunogenicor antigenic epitopes of the polypeptides described herein. An“immunogenic epitope” is defined as a part of a protein that elicits anantibody response when the whole protein is the immunogen. On the otherhand, a region of a protein molecule to which an antibody can bind isdefined as an “antigenic epitope.” The number of immunogenic epitopes ofa protein generally is less than the number of antigenic epitopes. See,for instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002(1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A. (1983) Antibodies that react withpredetermined sites on proteins. Science 219:660-666. Peptides capableof eliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777. Antigenicepitope-bearing peptides and polypeptides of the invention preferablycontain a sequence of at least seven, more preferably at least nine andmost preferably between at least about 15 to about 30 amino acidscontained within the amino acid sequence of a polypeptide of theinvention.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate TR2 receptor-specific antibodies include: a polypeptidecomprising amino acid residues from about 39 to about 70 in FIG. 1(amino acid residues 3 to 34 in SEQ ID NO:2); a polypeptide comprisingamino acid residues from about 106 to about 120 in FIG. 1A-1B (aminoacid residues 70 to 84 in SEQ ID NO:2); a polypeptide comprising aminoacid residues from about 142 to about 189 in FIG. 1A-1B (amino acidresidues 106 to 153 in SEQ ID NO:2); a polypeptide comprising amino acidresidues from about 276 to about 283 in FIG. 1 (amino acid residues 240to 247 in SEQ ID NO:2); a polypeptide comprising amino acid residuesfrom about 39 to about 70 in FIG. 4A-4C (amino acid residues 3 to 34 inSEQ ID NO:5); a polypeptide comprising amino acid residues from about 99to about 136 in FIG. 4A-4C (amino acid residues 63 to 100 in SEQ IDNO:5); a polypeptide comprising amino acid residues from about 171 toabout 185 in FIG. 4A-4C (amino acid residues 135 to 149 in SEQ ID NO:5);a polypeptide comprising amino acid residues from about 56 to about 68in FIG. 7A-7C (SEQ ID NO:8); and a polypeptide comprising amino acidresidues from about 93 to about 136 in FIG. 7A-7C (SEQ ID NO:8). Asindicated above, the inventors have determined that the abovepolypeptide fragments are antigenic regions of the TR2 receptorproteins.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A. (1985) Generalmethod for the rapid solid-phase synthesis of large numbers of peptides:specificity of antigen-antibody interaction at the level of individualamino acids. Proc. Natl. Acad. Sci. USA 82:5131-5135. This “SimultaneousMultiple Peptide Synthesis (SMPS)” process is further described in U.S.Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, TR2 polypeptides of thepresent invention and the epitope-bearing fragments thereof describedabove can be combined with parts of the constant domain ofimmunoglobulins (IgG), resulting in chimeric polypeptides. These fusionproteins facilitate purification and show an increased half-life invivo. This has been shown, e.g., for chimeric proteins consisting of thefirst two domains of the human CD4-polypeptide and various domains ofthe constant regions of the heavy or light chains of mammalianimmunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86(1988)). Fusion proteins that have a disulfide-linked dimeric structuredue to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TR2 receptor proteins orprotein fragments alone (Fountoulakis et al., J. Biochem 270:3958-3964(1995)).

Detection of Disease States

The TNF-family ligands induce various cellular responses by binding toTNF-family receptors, including the TR2 receptors of the presentinvention. TNF-β, a potent ligand of the TNF receptor proteins, is knownto be involved in a number of biological processes including lymphocytedevelopment, tumor necrosis, induction of an antiviral state, activationof polymorphonuclear leukocytes, induction of class I majorhistocompatibility complex antigens on endothelial cells, induction ofadhesion molecules on endothelium and growth hormone stimulation (Ruddleand Homer, Prog. Allergy, 40:162-182 (1988)). TNF-α, also a ligand ofthe TNF receptor proteins, has been reported to have a role in the rapidnecrosis of tumors, immunostimulation, autoimmune disease, graftrejection, producing an anti-viral response, septic shock, cerebralmalaria, cytotoxicity, protection against deleterious effects ofionizing radiation produced during a course of chemotherapy, such asdenaturation of enzymes, lipid peroxidation and DNA damage (Nata et al,J. Immunol. 136(7):2483 (1987); Porter, Tibtech 9:158-162 (1991)),growth regulation, vascular endothelium effects and metabolic effects.TNF-α also triggers endothelial cells to secrete various factors,including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation. Inaddition, TNF-α up-regulates various cell adhesion molecules such asE-Selectin, ICAM-1 and VCAM-1. TNF-α and the Fas ligand have also beenshown to induce programmed cell death.

Cells which express the TR2 polypeptides and are believed to have apotent cellular response to TR2 receptor ligands include B lymphocytes(CD19⁺), both CD4⁺ and CD8⁺ T lymphocytes, monocytes, endothelial cellsand other cell types shown in Tables 2 and 3. By “a cellular response toa TNF-family ligand” is intended any genotypic, phenotypic, and/ormorphologic change to a cell, cell line, tissue, tissue culture orpatient that is induced by a TNF-family ligand. As indicated, suchcellular responses include not only normal physiological responses toTNF-family ligands, but also diseases associated with increased cellproliferation or the inhibition of increased cell proliferation, such asby the inhibition of apoptosis. Apoptosis—programmed cell death—is aphysiological mechanism involved in the deletion of peripheral Tlymphocytes of the immune system, and its dysregulation can lead to anumber of different pathogenic processes (Ameisen, J. C., AIDS8:1197-1213 (1994); Krammer, P. H. et al., Curr. Opin. Immunol.6:279-289 (1994)).

It is believed that certain tissues in mammals with specific diseasestates associated with aberrant cell survival express significantlyaltered levels of the TR2 receptor protein and mRNA encoding the TR2receptor protein when compared to a corresponding “standard” mammal,i.e., a mammal of the same species not having the disease state.Further, since some forms of this protein are secreted, it is believedthat enhanced levels of the TR2 receptor protein can be detected incertain body fluids (e.g., sera, plasma, urine, and spinal fluid) frommammals with the disease state when compared to sera from mammals of thesame species not having the disease state. Thus, the invention providesa diagnostic method useful during diagnosis of disease states, whichinvolves assaying the expression level of the gene encoding the TR2receptor protein in mammalian cells or body fluid and comparing the geneexpression level with a standard TR2 receptor gene expression level,whereby an increase or decrease in the gene expression level over thestandard is indicative of certain disease states associated withaberrant cell survival.

Where diagnosis of a disease state involving the TR2 receptors of thepresent invention has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting significantly aberrant TR2 receptor geneexpression will experience a worse clinical outcome relative to patientsexpressing the gene at a lower level.

By “assaying the expression level of the gene encoding the TR2 receptorprotein” is intended qualitatively or quantitatively measuring orestimating the level of the TR2 receptor protein or the level of themRNA encoding the TR2 receptor protein in a first biological sampleeither directly (e.g., by determining or estimating absolute proteinlevel or mRNA level) or relatively (e.g., by comparing to the TR2receptor protein level or mRNA level in a second biological sample).

Preferably, the TR2 receptor protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardTR2 receptor protein level or mRNA level, the standard being taken froma second biological sample obtained from an individual not having thedisease state. As will be appreciated in the art, once a standard TR2receptor protein level or mRNA level is known, it can be used repeatedlyas a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source which containsTR2 receptor protein or mRNA. Biological samples include mammalian bodyfluids (such as sera, plasma, urine, synovial fluid and spinal fluid)which contain secreted mature TR2 receptor protein, and thymus,prostate, heart, placenta, muscle, liver, spleen, lung, kidney and othertissues. Methods for obtaining tissue biopsies and body fluids frommammals are well known in the art. Where the biological sample is toinclude mRNA, a tissue biopsy is the preferred source.

Diseases associated with increased cell survival, or the inhibition ofapoptosis, include cancers (such as follicular lymphomas, carcinomaswith p53 mutations, and hormone-dependent tumors); autoimmune disorders(such as systemic lupus erythematosus and immune-relatedglomerulonephritis rheumatoid arthritis) and viral infections (such asherpes viruses, pox viruses and adenoviruses), information graft v. hostdisease, acute graft rejection, and chronic graft rejection. Diseasesassociated with decreased cell survival, or increased apoptosis, includeAIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such asaplastic anemia), ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), toxin-induced liver disease(such as that caused by alcohol), septic shock, cachexia and anorexia.

Assays available to detect levels of soluble receptors are well known tothose of skill in the art, for example, radioimmunoassays,competitive-binding assays, Western blot analysis, and preferably anELISA assay may be employed.

TR2 receptor-protein specific antibodies can be raised against theintact TR2 receptor protein or an antigenic polypeptide fragmentthereof, which may presented together with a carrier protein, such as analbumin, to an animal system (such as rabbit or mouse) or, if it is longenough (at least about 25 amino acids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (mAb)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)₂ fragments) which are capable ofspecifically binding to TR2 receptor protein. Fab and F(ab′)₂ fragmentslack the Fc fragment of intact antibody, clear more rapidly from thecirculation, and may have less non-specific tissue binding of an intactantibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus, thesefragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the TR2 receptorprotein or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of TR2 receptor proteinis prepared and purified to render it substantially free of naturalcontaminants. Such a preparation is then introduced into an animal inorder to produce polyclonal antisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or TR2 receptor protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J.Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., (1981) pp. 563-681). In general, such procedures involveimmunizing an animal (preferably a mouse) with a TR2 receptor proteinantigen or, more preferably, with a TR2 receptor protein-expressingcell. Suitable cells can be recognized by their capacity to bindanti-TR2 receptor protein antibody. Such cells may be cultured in anysuitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56° C.), and supplemented with about10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, andabout 100 μg/ml of streptomycin. The splenocytes of such mice areextracted and fused with a suitable myeloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; however, it is preferable to employ the parent myeloma cellline (SP₂O), available from the American Type Culture Collection,Manassas, Va. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al. (Gastroenterology 80:225-232(1981)). The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingthe TR2 receptor protein antigen.

Agonists and Antagonists of TR2Receptor Function

In one aspect, the present invention is directed to a method forinhibiting an activity of TR2 induced by a TNF-family ligand (e.g., cellproliferation, hematopoietic development), which involves administeringto a cell which expresses a TR2 polypeptide an effective amount of a TR2receptor ligand, analog or an antagonist capable of decreasing TR2,receptor mediated signaling. Preferably, TR2 receptor mediated signalingis increased to treat a disease wherein increased cell proliferation isexhibited. An antagonist can include soluble forms of the TR2 receptorsand antibodies directed against the TR2 polypeptides which block TR2receptor mediated signaling. Preferably, TR2 receptor mediated signalingis decreased to treat a disease.

In a further aspect, the present invention is directed to a method forincreasing cell proliferation induced by a TNF-family ligand, whichinvolves administering to a cell which expresses a TR2 polypeptide aneffective amount of an agonist capable of increasing TR2 receptormediated signaling. Preferably, TR2 receptor mediated signaling isincreased to treat a disease wherein decreased cell proliferation isexhibited. Agonists of the present invention include monoclonalantibodies directed against the TR2 polypeptides which stimulate TR2receptor mediated signaling. Preferably, TR2 receptor mediated signalingis increased to treat a disease.

By “agonist” is intended naturally occurring and synthetic compoundscapable of enhancing cell proliferation and differentiation mediated byTR2 polypeptides. Such agonists include agents which increase expressionof TR2 receptors or increase the sensitivity of the expressed receptor.By “antagonist” is intended naturally occurring and synthetic compoundscapable of inhibiting TR2 mediated cell proliferation anddifferentiation. Such antagonists include agents which decreaseexpression of TR2 receptors or decrease the sensitivity of the expressedreceptor. Whether any candidate “agonist” or “antagonist” of the presentinvention can enhance or inhibit cell proliferation and differentiationcan be determined using art-known TNF-family ligand/receptor cellularresponse assays, including those described in more detail below.

One such screening technique involves the use of cells which express thereceptor (for example, transfected CHO cells) in a system which measuresextracellular pH changes caused by receptor activation, for example, asdescribed in Science 246:181-296 (October 1989). For example, compoundsmay be contacted with a cell which expresses the receptor polypeptide ofthe present invention and a second messenger response, e.g., signaltransduction or pH changes, may be measured to determine whether thepotential compound activates or inhibits the receptor.

Another such screening technique involves introducing RNA encoding thereceptor into Xenopus oocytes to transiently express the receptor. Thereceptor oocytes may then be contacted with the receptor ligand and acompound to be screened, followed by detection of inhibition oractivation of a calcium signal in the case of screening for compoundswhich are thought to inhibit activation of the receptor.

Another method involves screening for compounds which inhibit activationof the receptor polypeptide of the present invention antagonists bydetermining inhibition of binding of labeled ligand to cells which havethe receptor on the surface thereof. Such a method involves transfectinga eukaryotic cell with DNA encoding the receptor such that the cellexpresses the receptor on its surface and contacting the cell with acompound in the presence of a labeled form of a known ligand. The ligandcan be labeled, e.g., by radioactivity. The amount of labeled ligandbound to the receptors is measured, e.g., by measuring radioactivity ofthe receptors. If the compound binds to the receptor as determined by areduction of labeled ligand which binds to the receptors, the binding oflabeled ligand to the receptor is inhibited.

Soluble forms of the polypeptides of the present invention may beutilized in the ligand binding assay described above. These forms of theTR2 receptors are contacted with ligands in the extracellular mediumafter they are secreted. A determination is then made as to whether thesecreted protein will bind to TR2 receptor ligands.

Further screening assays for agonist and antagonist of the presentinvention are described in Tartaglia, L. A., and Goeddel, D. V., J.Biol. Chem. 267(7):4304-4307 (1992).

Thus, in a further aspect, a screening method is provided fordetermining whether a candidate agonist or antagonist is capable ofenhancing or inhibiting a cellular response to a TNF-family ligand. Themethod involves contacting cells which express TR2 polypeptides with acandidate compound and a TNF-family ligand, assaying a cellularresponse, and comparing the cellular response to a standard cellularresponse, the standard being assayed when contact is made with theligand in absence of the candidate compound, whereby an increasedcellular response over the standard indicates that the candidatecompound is an agonist of the ligand/receptor signaling pathway and adecreased cellular response compared to the standard indicates that thecandidate compound is an antagonist of the ligand/receptor signalingpathway. By “assaying a cellular response” is intended qualitatively orquantitatively measuring a cellular response to a candidate compoundand/or a TNF-family ligand (e.g., determining or estimating an increaseor decrease in T cell proliferation or tritiated thymidine labeling). Bythe invention, a cell expressing a TR2 polypeptide can be contacted witheither an endogenous or exogenously administered TNF-family ligand.

In an additional aspect, a thymocyte proliferation assay may be employedto identify both ligands and potential drug candidates. For example,thymus cells are disaggregated from tissue and grown in culture medium.Incorporation of DNA precursors such as ³H-thymidine or5-bromo-2′-deoxyuridine (BrdU) is monitored as a parameter for DNAsynthesis and cellular proliferation. Cells which have incorporated BrdUinto DNA can be detected using a monoclonal antibody against BrdU andmeasured by an enzyme or fluorochrome-conjugated second antibody. Thereaction is quantitated by fluorimetry or by spectrophotometry. Twocontrol wells and an experimental well are set up as above and TNF-β orcognate ligand is added to all wells while soluble receptor polypeptidesof the present invention are added individually to the second controlwells, with the experimental well containing a compound to be screened.The ability of the compound to be screened to stimulate or inhibit theabove interaction may then be quantified.

Agonists according to the present invention include compounds such as,for example, TNF-family ligand peptide fragments, transforming growthfactor β, and neurotransmitters (such as glutamate, dopamine,N-methyl-D-aspartate). Preferred agonist include polyclonal andmonoclonal antibodies raised against TR2 polypeptide, or a fragmentthereof. Such agonist antibodies raised against a TNF-family receptorare disclosed in Tartaglia, L. A., et al., Proc. Natl. Acad. Sci. USA88:9292-9296 (1991); and Tartaglia, L. A., and Goeddel, D. V., J. Biol.Chem. 267 (7):4304-4307 (1992). See, also, PCT Application WO 94/09137.Further preferred agonists include chemotherapeutic drugs such as, forexample, cisplatin, doxorubicin, bleomycin, cytosine arabinoside,nitrogen mustard, methotrexate and vincristine. Others include ethanoland β-amyloid peptide. (Science 267:1457-1458 (1995)).

Antagonist according to the present invention include soluble forms ofthe TR2 receptors (e.g., fragments of the TR2 receptor shown in FIG.1A-1B that include the ligand binding domain from the extracellularregion of the full length receptor). Such soluble forms of the receptor,which may be naturally occurring or synthetic, antagonize TR2, TR2-SV1or TR2-SV2 mediated signaling by competing with the cell surface boundforms of the receptor for binding to TNF-family ligands. Antagonists ofthe present invention also include antibodies specific for TNF-familyligands and TR2-Fc fusion proteins such as the one described below inExamples 5 and 6.

By a “TNF-family ligand” is intended naturally occurring, recombinant,and synthetic ligands that are capable of binding to a member of the TNFreceptor family and inducing the ligand/receptor signaling pathway.Members of the TNF ligand family include, but are not limited to, TNF-α,lymphotoxin-α (LT-α, also known as TNF-β), LT-β (found in complexheterotrimer LT-α2-β), FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L andnerve growth factor (NGF).

The experiments set forth in Example 6 demonstrate that the TR2receptors of the present invention are capable of inducing theproliferation of lymphocytes. Further, such proliferation can beinhibited by a TR2 protein fragment fused to an Fc antibody fragment.

TNF-α has been shown to protect mice from infection with herpes simplexvirus type 1 (HSV-1). Rossol-Voth, R. et al., J. Gen. Virol. 72:143-147(1991). The mechanism of the protective effect of TNF-α is unknown butappears to involve neither interferons not NK cell killing. One memberof the TNFR family has been shown to mediate HSV-1 entry into cells.Montgomery, R. et al., Eur. Cytokine Newt. 7:159 (1996). Further,antibodies specific for the extracellular domain of this TNFR blockHSV-1 entry into cells. Thus, TR2 receptors of the present inventioninclude both TR2 amino acid sequences and antibodies capable ofpreventing TNFR mediated viral entry into cells. Such sequences andantibodies can function by either competing with cell surface localizedTNFR for binding to virus or by directly blocking binding of virus tocell surface receptors.

Antibodies according to the present invention may be prepared by any ofa variety of methods using TR2 receptor immunogens of the presentinvention. Such TR2 receptor immunogens include the TR2 receptor proteinshown in FIG. 1A-1B (SEQ ID NO:2) and the TR2-SV1 (FIG. 4A-4C (SEQ IDNO:5)) and TR2-SV2 (FIG. 7A-7C (SEQ ID NO:8)) polypeptides (any of whichmay or may not include a leader sequence) and polypeptide fragments ofthe receptors comprising the ligand binding, extracellular,transmembrane, the intracellular domains of the TR2 receptors, or anycombination thereof.

Polyclonal and monoclonal antibody agonist or antagonist according tothe present invention can be raised according to the methods disclosedin Tartaglia and Goeddel, J. Biol. Chem. 267(7):4304-4307 (1992));Tartaglia et al., Cell 73:213-216 (1993)), and PCT Application WO94/09137. The term “antibody” (Ab) or “monoclonal antibody” (mAb) asused herein is meant to include intact molecules as well as fragmentsthereof (such as, for example, Fab and F(ab′)₂ fragments) which arecapable of binding an antigen. Fab and F(ab′)₂ fragments lack the Fcfragment of intact antibody, clear more rapidly from the circulation,and may have less non-specific tissue binding of an intact antibody(Wahl et al., J. Nucl. Med. 24:316-325 (1983)).

In a preferred method, antibodies according to the present invention aremAbs. Such mAbs can be prepared using hybridoma technology (Kohler andMillstein, Nature 256:495-497 (1975) and U.S. Pat. No. 4,376,110; Harlowet al., Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1988; Monoclonal Antibodies andHybridomas: A New Dimension in Biological Analyses, Plenum Press, NewYork, N.Y., 1980; Campbell, “Monoclonal Antibody Technology,” In:Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13(Burdon et al., eds.), Elsevier, Amsterdam (1984)).

Proteins and other compounds which bind the TR2 receptor domains arealso candidate agonist and antagonist according to the presentinvention. Such binding compounds can be “captured” using the yeasttwo-hybrid system (Fields and Song, Nature 340:245-246 (1989)). Amodified version of the yeast two-hybrid system has been described byRoger Brent and his colleagues (Gyuris, J. et al., Cell 75:791-803(1993); Zervos, A. S. et al., Cell 72:223-232 (1993)). Preferably, theyeast two-hybrid system is used according to the present invention tocapture compounds which bind to the ligand binding, extracellular,intracellular, and transmembrane domains of the TR2 receptors. Suchcompounds are good candidate agonist and antagonist of the presentinvention.

Using the two-hybrid assay described above, the intracellular domain ofthe TR2 receptor, or a portion thereof, may be used to identify cellularproteins which interact with the receptor in vivo. Such an assay mayalso be used to identify ligands with potential agonistic orantagonistic activity of TR2 receptor function. This screening assay haspreviously been used to identify protein which interact with thecytoplasmic domain of the murine TNF-RII and led to the identificationof two receptor associated proteins. Rothe, M. et al., Cell 78:681(1994). Such proteins and amino acid sequences which bind to thecytoplasmic domain of the TR2 receptors are good candidate agonist andantagonist of the present invention.

Other screening techniques include the use of cells which express thepolypeptide of the present invention (for example, transfected CHOcells) in a system which measures extracellular pH changes caused byreceptor activation, for example, as described in Science, 246:181-296(1989). In another example, potential agonists or antagonists may becontacted with a cell which expresses the polypeptide of the presentinvention and a second messenger response, e.g., signal transduction maybe measured to determine whether the potential antagonist or agonist iseffective.

The TR2 receptor agonists may be employed to stimulate ligandactivities, such as inhibition of tumor growth and necrosis of certaintransplantable tumors. The agonists may also be employed to stimulatecellular differentiation, for example, T-cell, fibroblasts andhemopoietic cell differentiation. Agonists to the TR2 receptor may alsoaugment TR2's role in the host's defense against microorganisms andprevent related diseases (infections such as that from Listeriamonocytogenes) and Chlamidiae. The agonists may also be employed toprotect against the deleterious effects of ionizing radiation producedduring a course of radiotherapy, such as denaturation of enzymes, lipidperoxidation, and DNA damage.

Agonists to the receptor polypeptides of the present invention may beused to augment TNF's role in host defenses against microorganisms andprevent related diseases. The agonists may also be employed to protectagainst the deleterious effects of ionizing radiation produced during acourse of radiotherapy, such as denaturation of enzymes, lipidperoxidation, and DNA damage.

The agonists may also be employed to mediate an anti-viral response, toregulate growth, to mediate the immune response and to treatimmunodeficiencies related to diseases such as HIV by increasing therate of lymphocyte proliferation and differentiation.

The antagonists to the polypeptides of the present invention may beemployed to inhibit ligand activities, such as stimulation of tumorgrowth and necrosis of certain transplantable tumors. The antagonistsmay also be employed to inhibit cellular differentiation, for example,T-cell, fibroblasts and hemopoietic cell differentiation. Antagonistsmay also be employed to treat autoimmune diseases, for example, graftversus host rejection and allograft rejection, and T-cell mediatedautoimmune diseases such as AIDS. It has been shown that T-cellproliferation is stimulated via a type 2 TNF receptor. Accordingly,antagonizing the receptor may prevent the proliferation of T-cells andtreat T-cell mediated autoimmune diseases.

The state of immunodeficiency that defines AIDS is secondary to adecrease in the number and function of CD4⁺ T-lymphocytes. Recentreports estimate the daily loss of CD4⁺ T cells to be between 3.5×10⁷and 2×10⁹ cells (Wei X., et al., Nature 373:117-122 (1995)). One causeof CD4⁺ T cell depletion in the setting of HIV infection is believed tobe HIV-induced apoptosis. Indeed, HIV-induced apoptotic cell death hasbeen demonstrated not only in vitro but also, more importantly, ininfected individuals (Ameisen, J. C., AIDS 8:1197-1213 (1994); Finkel,T. H., and Banda, N. K., Curr. Opin. Immunol. 6:605-615 (1995);Muro-Cacho, C. A. et al., J. Immunol. 154:5555-5566 (1995)).Furthermore, apoptosis and CD4⁺ T-lymphocyte depletion is tightlycorrelated in different animal models of AIDS (Brunner, T., et al.,Nature 373:441-444 (1995); Gougeon, M. L., et al., AIDS Res. Hum.Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in thoseanimal models in which viral replication does not result in AIDS(Gougeon, M. L. et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)).Further data indicates that uninfected but primed or activated Tlymphocytes from HIV-infected individuals undergo apoptosis afterencountering the TNF-family ligand FasL. Using monocytic cell lines thatresult in death following HIV infection, it has been demonstrated thatinfection of U937 cells with HIV results in the de novo expression ofFasL and that FasL mediates HIV-induced apoptosis (Badley, A. D. et al.,J. Virol. 70:199-206 (1996)). Further the TNF-family ligand wasdetectable in uninfected macrophages and its expression was upregulatedfollowing HIV infection resulting in selective killing of uninfectedCD4⁺ T-lymphocytes (Badley, A. D et al., J. Virol. 70:199-206 (1996)).

As shown in Example 6, the TR2 receptor shown in FIG. 1A-1B is expressedin CD4⁺ T-lymphocytes and is capable of inducing lymphocyteproliferation. Thus, by the invention, a method for treating HIV⁺individuals is provided which involves administering an agonist of thepresent invention to increase the rate of proliferation anddifferentiation of CD4⁺ T-lymphocytes. Such agonists include agentscapable of inducing the expression of TR2 receptors (e.g., TNF-α, PMAand DMSO) or enhancing the signal of such receptors which induceslymphocyte proliferation and differentiation. Modes of administrationand dosages are discussed in detail below.

In rejection of an allograft, the immune system of the recipient animalhas not previously been primed to respond because the immune system forthe most part is only primed by environmental antigens. Tissues fromother members of the same species have not been presented in the sameway that, for example, viruses and bacteria have been presented. In thecase of allograft rejection, immunosuppressive regimens are designed toprevent the immune system from reaching the effector stage. However, theimmune profile of xenograft rejection may resemble disease recurrencemore that allograft rejection. In the case of disease recurrence, theimmune system has already been activated, as evidenced by destruction ofthe native islet cells. Therefore, in disease recurrence the immunesystem is already at the effector stage. Antagonists of the presentinvention are able to suppress the immune response to both allograftsand xenografts by decreasing the rate of TR2 mediated lymphocyteproliferation and differentiation. Such antagonists include the TR2-Fcfusion protein described in Examples 5 and 6. Thus, the presentinvention further provides a method for suppression of immune responses.

In addition, TNF-α has been shown to prevent diabetes in strains ofanimals which are prone to this affliction resulting from autoimmunity.See Porter, A., Tibtech 9:158-162 (1991). Thus, agonists and antagonistsof the present invention may be useful in the treatment of autoimmunediseases such as type 1 diabetes.

In addition, the role played by the TR2 receptors in cell proliferationand differentiation indicates that agonist or antagonist of the presentinvention may be used to treat disease states involving aberrantcellular expression of these receptors. TR2 receptors may in somecircumstances induce an inflammatory response, and antagonists may beuseful reagents for blocking this response. Thus TR2 receptorantagonists (e.g., soluble forms of the TR2 receptors; neutralizingantibodies) may be useful for treating inflammatory diseases, such asrheumatoid arthritis, osteoarthritis, psoriasis, septicemia, andinflammatory bowel disease.

Antagonists to the TR2 receptor may also be employed to treat and/orprevent septic shock, which remains a critical clinical condition.Septic shock results from an exaggerated host response, mediated byprotein factors such as TNF and IL-1, rather than from a pathogendirectly. For example, lipopolysaccharides have been shown to elicit therelease of TNF leading to a strong and transient increase of its serumconcentration. TNF causes shock and tissue injury when administered inexcessive amounts. Accordingly, it is believed that antagonists to theTR2 receptor will block the actions of TNF and treat/prevent septicshock. These antagonists may also be employed to treat meningococcemiain children which correlates with high serum levels of TNF.

Among other disorders which may be treated by the antagonists to TR2receptors, there are included, inflammation which is mediated by TNFreceptor ligands, and the bacterial infections cachexia and cerebralmalaria. The TR2 receptor antagonists may also be employed to treatinflammation mediated by ligands to the receptor such as TNF.

Modes of Administration

The agonist or antagonists described herein can be administered invitro, ex vivo, or in vivo to cells which express the receptor of thepresent invention. By administration of an “effective amount” of anagonist or antagonist is intended an amount of the compound that issufficient to enhance or inhibit a cellular response to a TNF-familyligand and include polypeptides. In particular, by administration of an“effective amount” of an agonist or antagonists is intended an amounteffective to enhance or inhibit TR2 receptor mediated activity. Ofcourse, where cell proliferation and/or differentiation is to beenhanced, an agonist according to the present invention can beco-administered with a TNF-family ligand. One of ordinary skill willappreciate that effective amounts of an agonist or antagonist can bedetermined empirically and may be employed in pure form or inpharmaceutically acceptable salt, ester or pro-drug form. The agonist orantagonist may be administered in compositions in combination with oneor more pharmaceutically acceptable excipients.

It will be understood that, when administered to a human patient, thetotal daily usage of the compounds and compositions of the presentinvention will be decided by the attending physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular patient will depend upon factors well known inthe medical arts.

As a general proposition, the total pharmaceutically effective amount ofa TR2 polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the TR2 polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

Pharmaceutical compositions containing the TR2 receptor polypeptides ofthe invention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

Example 1 Expression and Purification of TR2 in E. Coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6×His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6×His tag.

The DNA sequence encoding the desired portion of the TR2 protein lackingthe hydrophobic leader sequence is amplified from the deposited cDNAclone using PCR oligonucleotide primers which anneal to the aminoterminal sequences of the desired portion of the TR2 protein and tosequences in the deposited construct 3′ to the cDNA coding sequence.Additional nucleotides containing restriction sites to facilitatecloning in the pQE60 vector are added to the 5′ and 3′ sequences,respectively.

For cloning the mature protein, the 5′ primer has the sequence: 5′CGCCCATGGCCCCAGCTCTGCCGTCCT 3′ (SEQ ID NO:14) containing the underlinedNcoI restriction site followed by 18 nucleotides complementary to theamino terminal coding sequence of the mature TR2 sequence in FIG. 1A-1B.One of ordinary skill in the art would appreciate, of course, that thepoint in the protein coding sequence where the 5′ primer begins may bevaried to amplify a desired portion of the complete protein shorter orlonger than the mature form. The 3′ primer has the sequence: 5′CGCAAGCTTATTGTGGGAGCTGCTGGTCCC 3′ (SEQ ID NO:15) containing theunderlined HindIII restriction site followed by 18 nucleotidescomplementary to the 3′ end of the nucleotide sequence shown in FIG.1A-1B (SEQ ID NO: 1) encoding the extracellular domain of the TR2receptor.

The amplified TR2DNA fragments and the vector pQE60 are digested withNcoI and HindIII and the digested DNAs are then ligated together.Insertion of the TR2DNA into the restricted pQE60 vector places the TR2protein coding region including its associated stop codon downstreamfrom the IPTG-inducible promoter and in-frame with an initiating AUG.The associated stop codon prevents translation of the six histidinecodons downstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing TR2 protein, is available commercially from QIAGEN, Inc.,supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4° C. in 6 M guanidine-HCl,pH 8. The cell debris is removed by centrifugation, and the supernatantcontaining the TR2 is dialyzed against 50 mM Na-acetate buffer pH 6,supplemented with 200 mM NaCl. Alternatively, the protein can besuccessfully refolded by dialyzing it against 500 mM NaCl, 20% glycerol,25 mM Tris/HCl pH 7.4, containing protease inhibitors. Afterrenaturation the protein can be purified by ion exchange, hydrophobicinteraction and size exclusion chromatography. Alternatively, anaffinity chromatography step such as an antibody column can be used toobtain pure TR2 protein. The purified protein is stored at 4° C. orfrozen at −80° C.

Example 2 Example 2(a) Cloning and Expression of a Soluble Fragment ofTR2Protein in a Baculovirus Expression System

In this example, the plasmid shuttle vector pA2 GP was used to insertthe cloned DNA encoding the mature extracellular domain of the TR2receptor protein shown in FIG. 1A-1B, lacking its naturally associatedsecretory signal (leader) sequence, into a baculovirus. This protein wasexpressed using a baculovirus leader and standard methods as describedin Summers et al., A Manual of Methods for Baculovirus Vectors andInsect Cell Culture Procedures, Texas Agricultural Experimental StationBulletin No. 1555 (1987). This expression vector contains the strongpolyhedrin promoter of the Autographa californica nuclear polyhedrosisvirus (AcMNPV) followed by the secretory signal peptide (leader) of thebaculovirus gp67 protein and convenient restriction sites such as BamHI,XbaI and Asp718. The polyadenylation site of the simian virus 40(“SV40”) is used for efficient polyadenylation. For easy selection ofrecombinant virus, the plasmid contains the beta-galactosidase gene fromE. coli under control of a weak Drosophila promoter in the sameorientation, followed by the polyadenylation signal of the polyhedringene. The inserted genes are flanked on both sides by viral sequencesfor cell-mediated homologous recombination with wild-type viral DNA togenerate viable virus that expresses the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39.

The cDNA sequence encoding essentially the mature extracellular domain(amino acids 37 to 200 shown in FIG. 1A-1B) of the TR2 receptor proteinin the deposited clone (ATCC™ Deposit Number 97059) was amplified usingPCR oligonucleotide primers corresponding to the relevant 5′ and 3′sequences of the gene. The 5′ primer for each of the above has thesequence: 5′ CGCGGATCCCGGAGCCCCCTGCTAC 3′ (SEQ ID NO: 16) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 15 bases of the codingsequence of the TR2 protein shown in FIG. 1A-1B, beginning with thenucleotide 354. The 3′ primer has the sequence:

5′ CGCGGTACCATTGTGGGAGCTGCTGGTCCC 3′ (SEQ ID NO:17) containing theunderlined, Asp718 restriction sites followed by 17 nucleotidescomplementary to the coding sequences in FIG. 1A-1B.

The amplified fragment was isolated from a 1% agarose gel using acommercially available kit (“GENECLEAN™,” BIO 101 Inc., La Jolla,Calif.). The fragment was then digested with BamHI and Asp718 andpurified on a 1% agarose gel. This fragment is designated herein F1″.

The plasmid was digested with the restriction enzymes BamHI and Asp718dephosphorylated using calf intestinal phosphatase. The DNA was thenisolated from a 1% agarose gel using a commercially available kit(“GENECLEAN™” BIO 101 Inc., La Jolla, Calif.). This vector DNA wasdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 were ligated togetherwith T4 DNA ligase. E. coli HB101 cells were transformed with theligation mixture and spread on culture plates. Other suitable E. colihosts such as XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.)may also be used. Bacteria were identified that contain the plasmid withthe human TR2 sequences using the PCR method, in which one of theprimers that was used to amplify the gene and the second primer was fromwell within the vector so that only those bacterial colonies containingTR2 gene fragments show amplification of the DNA. The sequence of thecloned fragment was confirmed by DNA sequencing. The plasmid wasdesignated herein pBacTR2-T.

Five μg of pBacTR2-T was co-transfected with 1.0 μg of a commerciallyavailable linearized baculovirus DNA (“BACULOGOLD™ baculovirus DNA”,Pharmingen, San Diego, Calif.), using the lipofection method describedby Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417 (1987). 1 μgof BACULOGOLD™ virus DNA and 5 μg of plasmid pBacTR2-T were mixed in asterile well of a microtiter plate containing 50 μl of serum-freeGrace's medium (Life Technologies Inc., Gaithersburg, Md.). Afterwards,10 μl LIPOFECTIN™ plus 90 μl Grace's medium were added, mixed andincubated for 15 minutes at room temperature. Then the transfectionmixture was added drop-wise to Sf9 insect cells (ATCC™ CRL 1711) seededin a 35 mm tissue culture plate with 1 ml Grace's medium without serum.The plate was rocked back and forth to mix the newly added solution. Theplate was then incubated for 5 hours at 27° C. After 5 hours thetransfection solution was removed from the plate and 1 ml of Grace'sinsect medium supplemented with 10% fetal calf serum was added. Theplate was put back into an incubator and cultivation was continued at27° C. for four days.

After four days the supernatant was collected and a plaque assay wasperformed, as described by Summers and Smith, supra. An agarose gel with“BLUE GAL™” (Life Technologies Inc., Gaithersburg) was used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, page 9-10). After appropriate incubation, blue stainedplaques were picked with the tip of a micropipettor (e.g., Eppendorf).The agar containing the recombinant viruses was then resuspended in amicrocentrifuge tube containing 200 μl of Grace's medium and thesuspension containing the recombinant baculovirus was used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes were harvested and then they were stored at 4° C. Therecombinant virus is called V-TR2-T.

To verify the expression of the gene used, Sf9 cells were grown inGrace's medium supplemented with 10% heat inactivated FBS. The cellswere infected with the recombinant baculovirus V-TR2-T at a multiplicityof infection (“MOI”) of about 2. Six hours later the medium was removedand replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). Forty-two hourslater, 5 μCi of ³⁵S-methionine and 5 μCi ³⁵S-cysteine (available fromAmersham) were added to radiolabel proteins. The cells were furtherincubated for 16 hours and then they were harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinswere analyzed by SDS-PAGE followed by autoradiography. Microsequencingof the amino acid sequence of the amino terminus of purified protein wasused to determine the amino terminal sequence of the mature protein andthus the cleavage point and length of the secretory signal peptide.

Example 2(b) Cloning and Expression of TR2Protein in a BaculovirusExpression System

Similarly to the cloning and expression of the truncated version of theTR2 receptor described in Example 2(a), recombinant baculoviruses weregenerated which express the full length TR2 receptor protein shown inFIG. 1A-1B (SEQ ID NO:2).

In this example, the plasmid shuttle vector pA2 was used to insert thecloned DNA encoding the complete protein, including its naturallyassociated secretary signal (leader) sequence, into a baculovirus toexpress the mature TR2 protein. Other attributes of the pA2 vector areas described for the pA2 GP vector used in Example 2(a).

The cDNA sequence encoding the full length TR2 protein in the depositedclone, including the AUG initiation codon and the naturally associatedleader sequence shown in FIG. 1A-1B (SEQ ID NO:2), was amplified usingPCR oligonucleotide primers corresponding to the 5′ and 3′ sequences ofthe gene. The 5′ primer has the sequence: 5′GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3′ (SEQ ID NO: 18) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 21 bases of thesequence of the complete TR2 protein shown in FIG. 1A-1B, beginning withthe AUG initiation codon. The 3′ primer has the sequence: 5′GCGCGGTACCTCTACCCCAGCAGGGGCGCCA 3′ (SEQ ID NO:19) containing theunderlined, Asp718 restriction site followed by 21 nucleotidescomplementary to the 3′ noncoding sequence in FIG. 1A-1B.

The amplified fragment was isolated and digested with restrictionenzymes as described in Example 2(a) to produce plasmid pBacTR2

5 μg of pBacTR2 was co-transfected with 1 μg of BACULOGOLD™ (Pharmingen)viral DNA and 10 μl of LIPOFECTIN™ (Life Technologies, Inc.) in a totalvolume of 200 μl serum free media. The primary viruses were harvested at4-5 days post-infection (pi), and used in plaque assays. Plaque purifiedviruses were subsequently amplified and frozen, as described in Example2(a).

For radiolabeling of expressed proteins, Sf9 cells were seeded in 12well dishes with 2.0 ml of a cell suspension containing 0.5×10⁶ cells/mland allowed to attach for 4 hours. Recombinant baculoviruses were usedto infect the cells at an MOI of 1-2. After 4 hours, the media wasreplaced with 1.0 ml of serum free media depleted for methionine andcysteine (-Met/-Cys). At 3 days pi, the culture media was replaced with0.5 ml-Met/-Cys containing 2 μCi each [³⁵S]-Met and [³⁵S]-Cys. Cellswere labeled for 16 hours after which the culture media was removed andclarified by centrifugation (Supernatant). The cells were lysed in thedish by addition of 0.2 ml lysis buffer (20 mM HEPES, pH 7.9; 130 mMNaCl; 0.2 mM EDTA; 0.5 mM DTT and 0.5% vol/vol NP-40) and then dilutedup to 1.0 ml with dH₂O (Cell Extract). 30 μl of each supernatant andcell extract were resolved by 15% SDS-PAGE. Protein gels were stained,destained, amplified, dried and autoradiographed. Labeled bandscorresponding to the recombinant proteins were visible after 16-72 hoursexposure.

Example 3 Cloning and Expression of TR2 in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC™ 37152), pSV2dhfr (ATCC™37146) and pBC12MI (ATCC™ 67109). Mammalian host cells that could beused include, human HeLa 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10: 169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology,438447 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart etal., Cell 41:521-530 (1985)). Multiple cloning sites, e.g., with therestriction enzyme cleavage sites BamHI, XbaI and Asp718, facilitate thecloning of the gene of interest. The vectors contain in addition the 3′intron, the polyadenylation and termination signal of the ratpreproinsulin gene.

Example 3(a) Cloning and Expression in COS Cells

The expression plasmid, pTR2HA, is made by cloning a cDNA encoding TR2into the expression vector pcDNAI/amp or pcDNAIII (which can be obtainedfrom Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding a TR2 is cloned into the polylinker region ofthe vector so that recombinant protein expression is directed by the CMVpromoter. The plasmid construction strategy is as follows. The TR2 cDNAof the deposited clone is amplified using primers that containconvenient restriction sites, much as described above for constructionof vectors for expression of TR2 in E. coli. Suitable primers includethe following, which are used in this example. The 5′ primer, containingthe underlined BamHI site, a Kozak sequence, an AUG start codon and 6additional codons of the 5′ coding region of the complete TR2 has thefollowing sequence: 5′ GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3′ (SEQ IDNO:20). The 3′ primer, containing the underlined XbaI site, a stopcodon, HA tag, and 19 bp of 3′ coding sequence has the followingsequence (at the 3′ end):

5′ GCGCTCTAGATCAAGCGTAGTCTGGGACGTCGT (SEQ ID NO:21)ATGGGTAGTGGTTTGGGCTCCTCCC 3′.

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the TR2-encoding fragment.

For expression of recombinant TR2, COS cells are transfected with anexpression vector, as described above, using DEAE-DEXTRAN, as described,for instance, in Sambrook et al., Molecular Cloning: a LaboratoryManual, Cold Spring Laboratory Press, Cold Spring Harbor, N.Y. (1989).Cells are incubated under conditions for expression of TR2 by thevector.

Expression of the TR2-HA fusion protein is detected by radiolabeling andimmunoprecipitation, using methods described in, for example Harlow etal., Antibodies: A Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1988). To this end, two daysafter transfection, the cells are labeled by incubation in mediacontaining ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3(b) Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of TR2 protein. Plasmid pC4 isa derivative of the plasmid pSV2-dhfr (ATCC™ Accession No. 37146). Theplasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., Kellems, R. M.,Bertino, J. R., and Schimke, R. T., 1978, J. Biol. Chem. 253:1357-1370,Hamlin, J. L. and Ma, C. 1990, Biochem. et Biophys. Acta, 1097:107-143,Page, M. J. and Sydenham, M. A. 1991, Biotechnology 9:64-68). Cellsgrown in increasing concentrations of MTX develop resistance to the drugby overproducing the target enzyme, DHFR, as a result of amplificationof the DHFR gene. If a second gene is linked to the DHFR gene, it isusually co-amplified and over-expressed. It is known in the art thatthis approach may be used to develop cell lines carrying more than 1,000copies of the amplified gene(s). Subsequently, when the methotrexate iswithdrawn, cell lines are obtained which contain the amplified geneintegrated into one or more chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen, et al., Molecular and Cellular Biology, March 1985:438-447)plus a fragment isolated from the enhancer of the immediate early geneof human cytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human M-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. CLONTECH'S™ Tet-Off and Tet-On geneexpression systems and similar systems can be used to express the TR2protein in a regulated way in mammalian cells (Gossen, M., & Bujard, H.1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551). For the polyadenylationof the mRNA other signals, e.g., from the human growth hormone or globingenes can be used as well. Stable cell lines carrying a gene of interestintegrated into the chromosomes can also be selected uponco-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHI andAsp718 and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete TR2 protein including its leadersequence is amplified using PCR oligonucleotide primers corresponding tothe 5′ and 3′ sequences of the gene. The 5′ primer has the sequence:

5′ GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3′ (SEQ ID NO:22) containing theunderlined BamHI restriction enzyme site followed by an efficient signalfor initiation of translation in eukaryotes, as described by Kozak, M.,J. Mol. Biol. 196:947-950 (1987), and 21 bases of the coding sequence ofTR2 protein shown in FIG. 1A-1B (SEQ ID NO:1). The 3′ primer has thesequence: 5′ GCGCGGTACCTCTACCCCAGCAGGGGCGCCA 3′ (SEQ ID NO:19)containing the underlined Asp718 restriction site followed by 21nucleotides complementary to the non-translated region of the TR2 geneshown in FIG. 1A-1B (SEQ ID NO: 1).

The amplified fragment is digested with the endonucleases BamHI andAsp718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. 5 μg of the expression plasmid pC4 is cotransfected with0.5 μg of the plasmid pSV2-neo using LIPOFECTIN™ (Felgner et al.,supra). The plasmid pSV2neo contains a dominant selectable marker, theneo gene from Tn5 encoding an enzyme that confers resistance to a groupof antibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 mM, 20 mM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4 Tissue Distribution of TR2 mRNA Expression

Northern blot analysis is carried out to examine TR2 gene expression inhuman tissues, using methods described by, among others, Sambrook etal., cited above. A cDNA probe containing the entire nucleotide sequenceof the TR2 protein (SEQ ID NO: 1) is labeled with ³²P using theREDIPRIME™ DNA labeling system (Amersham Life Science), according tomanufacturer's instructions. After labeling, the probe is purified usinga CHROMA SPIN-100 column (CLONTECH™ Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for TR2 mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from CLONTECH™ andare examined with the labeled probe using EXPRESSHYB™ hybridizationsolution (CLONTECH™) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70° C. overnight, and films developed according tostandard procedures.

Example 5 Example 5(a) Expression and Purification of TR2-Fc (TR2-IgFusion Protein) and Cleaved TR2

The putative transmembrane domain of translated TR2 receptor wasdetermined by hydrophobicity using the method of Goldman et al (Ann.Rev. of Biophys. Biophys. Chem. 15:321-353 (1986)) for identifyingnonpolar transbilayer helices. The region upstream of this transmembranedomain, encoding the putative leader peptide and extracellular domain,was chosen for the production of an Fc fusion protein. Primers weredesigned to PCR the corresponding coding region from HTXBS40 with theaddition of a Bg1II site (single underlined), a Factor Xa protease siteand an Asp718I site (double underlined) at the 3 end. PCR with thisprimer pair (forward 35-mer: 5′ CAGGAATTCGCAGCCATGGAGCCTCCTGGAGACTG 3′(SEQ ID NO:23), and reverse primer 53-mer: 5CCATACCCAGGTACCCCTTCCCTCGATAGATCT TGCCTTCGTCACCAGCCAGC 3 (SEQ IDNO:24)), which contains 18 nucleotides of the TR2 coding sequence,resulted in one band of the expected size. This was cloned intoCOSFclink to give the TR2-Fclink plasmid. The PCR product was digestedwith EcoRI and Asp718I and ligated into the COSFclink plasmid (Johansen,et al., J. Biol. Chem. 270:9459-9471 (1995)) to produce TR2-Fclink.

COS cells were transiently transfected with TR2-Fclink and the resultingsupernatant was immunoprecipitated with protein A agarose. Western blotanalysis of the immunoprecipitate using goat anti-human Fc antibodiesrevealed a strong band consistent with the expected size forglycosylated TR2-Fc (greater than 47.5 kD). A 15 L transient COStransfection was performed and the resulting supernatant was purified(see below). The purified protein was used to immunize mice followingDNA injection for the production of mAbs.

CHO cells were transfected with TR2-Fclink to produce stable cell lines.Five lines were chosen by dot blot analysis for expansion and wereadapted to shaker flasks. The line with the highest level of TR2-Fcprotein expression was identified by Western blot analysis. TR2-Fcprotein purified from the supernatant of this line was used for cellbinding studies by flow cytometry, either as intact protein or afterfactor Xa cleavage and biotinylation (see below).

Clone HTXBS40 is an allelic variant of TR2 which differs from thesequence shown in FIG. 1A-1B (SEQ ID NO: 1) in that HTXBS40 containsguanine at nucleotide 314, thymine at nucleotide 386 and cytosine atnucleotide 627.

A plasmid suitable for expression of the extracellular domain of TR2 wasconstructed as follows to immunize mice for the production of anti-TR2mAbs. The Fc fragment was removed from TR2-Fclink by a Bg1II/XbaIdigestion, Klenow was used to fill in the overhangs, and the blunt endsof the plasmid were religated. The resulting frame shift introduced astop codon immediately following the amino acids which had originallybeen introduced into TR2-Fclink by the addition of the Bg1II site. Thus,the C terminus of the extracellular domain of TR2 is followed by only 2amino acids (RS) in this constructed (TR2exlink).

Example 5(b) Purification of TR2-Fc from CHO E1A Conditioned MediaFollowed by Cleavage and Biotinylation of TR2

Assays

Product purity through the purification was monitored on 15% LaemmliSDS-PAGE gels run under reducing and non-reducing conditions. Proteinconcentration was monitored by A₂₈₀ assuming an extinction coefficientof 0.7 for the receptor and 1.28 for the chimera, both calculated fromthe sequence. Extinction coefficients were confirmed by AAA.

Protein G Chromatography of the TR2-Fc Fusion Protein

All steps described below were carried out at 4° C. 15 L of CHOconditioned media (CM) (0.2μ filtered following harvest in cell culture)was applied to a 5×10 cm column of Protein G at a linear flow rate of199 cm/h. The column had been washed with 100 mM glycine, pH 2.5 andequilibrated in 20 mM sodium phosphate, 150 mM sodium chloride, pH 7prior to sample application. After the CM was loaded the column waswashed with 5 column volumes of 20 mM sodium phosphate, 150 mM sodiumchloride, pH 7 and eluted with 100 mM glycine, pH 2.5. 435 ml of eluatewas immediately neutralized with 3 M Tris, pH 8.5 and 0.2μ filtered.Based on A, extinction coefficient 1.28, 65 mg of protein was recoveredat 0.15 mg/ml.

Concentration/Dialysis

385 ml of Protein G eluate was concentrated in an Amicon stirred cellfitted with a 30K membrane to 34 ml at a final concentration of 1.7. Theconcentrate was dialyzed against buffer.

Factor Xa Cleavage and Purification to Generate Free Receptor

Six ml (10.2 mg) of TR2-Fc was added to 50 μg of Factor Xa resulting ina 1:200, e:s ratio. The mixture was incubated overnight at 4° C.

Protein G Chromatography of the Free TR2 Receptor

A 1 ml column of Protein G was equilibrated in 20 mM sodium phosphate,150 mM sodium chloride, pH 6.5 in a disposable column using gravityflow. The cleaved receptor was passed over the column 3 times afterwhich the column was washed with 20 mM sodium phosphate, 150 mM sodiumchloride, pH 6.5 until no A absorbance was seen. The column was elutedwith 2.5 ml of 100 mM glycine, pH 2.5 neutralized with 83 μl of 3 MTris, pH 8.5. TR2 eluted in the nonbound fraction.

Concentration

The nonbound fraction from the Protein G column, about 12 ml, wasconcentrated in a Centricon 10K cell (Amicon) to about 1 ml to a finalconcentration of 3.5 mg/ml estimated by A, extinction coefficient 0.7.

Mono S Chromatography

The concentrated sample was diluted to 5 ml with 20 mM sodium phosphate,pH 6 and applied to a 0.5×5 cm Mono S column equilibrated in 20 mMsodium phosphate, pH 6 at a linear flow rate of 300 cm/h. The column waswashed with 20 mM sodium phosphate, pH 6 and eluted with a 20 columnvolume linear gradient of 20 mM sodium phosphate, pH 6 to 20 mM sodiumphosphate, 1 M sodium chloride, pH 6. TR2 protein eluted in the nonboundfraction.

Concentration/Dialysis

The 3 ml nonbound fraction from the Mono S column was concentrated to 1ml as above using a Centricon 10K cell and dialyze against 20 mM sodiumphosphate, 150 mM sodium chloride, pH 7. The concentration followingdialysis was 2.1 mg/ml.

Biotinylation

0.5 mg of TR2 at 2.1 mg/ml was dialyzed against 100 mM borate, pH 8.5. A20-fold molar excess of NHS-LC Biotin was added and the mixture was lefton a rotator overnight at 4° C. The biotinylated TR2 was dialyzedagainst. 20 mM sodium phosphate, 150 mM sodium chloride, pH 7, sterilefiltered and stored at −70° C. Biotinylation was demonstrated on aWestern blot probed with strepavidin HRP and subsequently developed withECL reagent.

Example 6 The Membrane Bound Form of the TR2 Receptor is a TNFR whichInduces Lymphocytes Proliferation and Differentiation

The members of the tumor necrosis factor (TNFR)/nerve growth factorreceptor (NGFR) superfamily are characterized by the presence of threeto six repeats of a cysteine-rich motif that consists of approximately30 to 40 amino acids in the extracellular part of the molecule (Mallett,S, and Barclay, A. N., Immunol. Today 12:220 (1991)). The crystalstructure of TNFR-I showed that the cysteine-rich motif (TNFR domain)was composed of three elongated strands of residues held together by atwisted ladder of disulfide bonds (Banner, D. W. et al., Cell 73:431(1993). These receptors contain a hinge-like region immediately adjacentto the transmembrane domain, characterized by a lack of cysteineresidues and a high proportion of serine, threonine, and proline, whichare likely to be glycosylated with O-linked sugars. A cytoplasmic partof these molecules shows limited sequence similarities—a finding whichmay be the basis for diverse cellular signaling. At present, the membersidentified from human cells include CD40 (Stamenkovic, I. et al., EMBOJ. 8:1403 (1989)), 4-1BB (Kwon, B. S, and Weissman, S. M., Proc. Natl.Acad. Sci. USA 86:1963 (1989)), OX-40 (Mallett, S. et al., EMBO J.9:1063 (1990)), TNFR-I (Loetscher, H. et al., Cell 61:351 (1990);Schall, T. J. et al., Cell 61:361 (1990)), TNFR-II (Smith, C. A. et al.,Science 248:1019 (1990)), CD27 (Van Lier, R. A. et al., J. Immunol.139:1589 (1987)), Fas (Itoh, N. et al., Cell 66:233 (1991)), NGFR(Johnson, D. et al., Cell 47:545 (1986)), CD30 (Durkop, H. et al., Cell68:421 (1992)) and LTBR (Baens, M. et al., Genomics 16:214 (1993)).Viral open reading frames encoding soluble TNFRs have also beenidentified, such as SFV-T2 (Smith, C. A. et al., Science 248:1019(1990)), Va53 (Howard, S. T. et al., Virology 180:633 (1991)), G4RG (Hu,F.-Q. et al., Virology 204:343 (1994)) and crmB (Smith, G. L., J. Gen.Viol 74:1725 (1993)).

Recent intensive studies have shown that these molecules are involved indiverse biological activities such as immunoregulation (Armitage, R. J.,Curr. Opin. Immunol. 6:407 (1994); Smith, C. A. et al., Cell 75:959(1994)), by regulating cell proliferation (Banchereau, J. et al.,Science 251:70 (1991); Pollok, K. E. et al., J. Immunol. 150:771 (1993);Baum, P. R. et al., EMBO J. 13:3992 (1994)), cell survival (Grass, H.-J.et al., Blood 83:2045 (1994); Torcia, M. et al., Cell 85:345-356(1996)), and cell death (Tartaglia, L. A. et al., Cell 74:845 (1993);Gillette-Ferguson, I. and Sidman, C. L., Eur. J. Immunol. 24:1181(1994); Krammer, P. H. et al., Curr. Opin. Immunol. 6:279 (1994)).

Because of their biological significance and the diverse membership ofthis superfamily, we predicted that there would be further members ofthe superfamily. By searching an EST-data base, we have identified a newmember of the TNFR superfamily. We report here the initialcharacterization of the molecule called TR2.

Material and Methods

Identification and Cloning of New Members of the TNFR Superfamily

An expressed sequence tag (EST) cDNA data base, obtained over 500different cDNA libraries (Adams, M. D. et al., Science 252:1651 (1991);Adams, M. D. et al., Nature 355:632 (1992)), was screened for sequencesimilarity with cysteine-rich motif of the TNFR superfamily, using theblastn and tblastn algorithms (Altschul, S. F. et al., J. Mol. Biol.215:403 (1990)). One EST (HT1SB52-ATCC™ Accession No. 97059) wasidentified in a human T cell line library which showed significantidentity to TNFR-II at the amino acid level. This sequence was used toclone the missing 5′ end by RACE (rapid amplification of cDNA ends)using a 5′-RACE-ready cDNA of human leukocytes (CLONTECH™, PT1155-1.Cat. #7301-1). This sequence matched four further ESTs (HTOBH42,HTOAU65, HLHA49 and HTXBS40). Complete sequencing of these and othercDNAs indicated that they contained an identical open reading framehomologous to the TNFR superfamily and was named TR2. Analysis ofseveral other ESTs and cDNAs indicated that some cDNAs had additionalsequences inserted in the open reading frame identified above, and mightrepresent various partially-spliced mRNAs.

Cells

The myeloid and B-cell lines studied represent cell types at differentstages of the differentiation pathway. KG1a and PLB 985 (Koeffler, H. etal., Blood 56:265 (1980); Tucker, K. et al., Blood 70:372 (1987)) wereobtained from Phillip Koeffler (UCLA School of Medicine), BJA-B was fromZ. Jonak (SmithKline Beecham), and TF 274, a stromal cell lineexhibiting osteoblastic features, was generated from the bone marrow ofa healthy male donor (Tan & Jonak, unpublished). All of the other celllines were obtained from the American Type Culture Collection (Manassas,Va.). Monocytes were prepared by differential centrifugation ofperipheral blood mononuclear cells (PBMC) and adhesion to tissue culturedish. CD19⁺, CD4⁺ and CD8⁺ were isolated from PBMC by immunomagneticbeads (Dynal, Lake Success, N.Y.). Endothelial cells from human coronaryartery were purchased from clonetics (Clonetics, Calif.).

RNA and DNA Blot Hybridization

Total RNA of adult tissues was purchases from CLONTECH™ (Palo Alto,Calif.), or extracted from primary cells and cell lines with TriReagent(Molecular Research Center, Inc., Cincinnati, Ohio). 5 to 7.5 μg oftotal RNA was fractionated in a 1% agarose gel containing formaldehyde,as described (Sambrook et al., Molecular Cloning, Cold Springs Harbor(1989)) and transferred quantitatively to Zeta-probe nylon membrane(Biorad, Hercules, Calif.) by vacuum-blotting. The blots wereprehybridized, hybridized with ³²P-labeled XhoI/EcoRI fragment of TR2 orOX-40 probe, washed under stringent conditions and exposed to X-rayfilms.

High molecular weight human DNA was digested with various restrictionenzymes and fractionated in 0.8% agarose gel. The DNA was denatured,neutralized and transferred to nylon membrane and hybridized to³²P-labeled TR-2 or its variant cDNA.

In Situ Hybridization and FISH Detection

The in situ hybridization and FISH detection of TR2 location in humanchromosome were performed as previously described (Heng, H. H. Q. etal., Proc. Natl. Acad. Sci. USA 89:9509 (1992); Heng, H. H. Q. et al.,Human Molecular Genetics 3:61 (1994)). FISH signals and the DAPI bandingpattern were recorded separately by taking photographs, and theassignment of the FISH mapping data with chromosomal bands was achievedby superimposing FISH signals with DAPI banded chromosome (Heng, H. H.Q. and Tsui, L.-C., Chromosoma. 102:325 (1993)).

Production of Recombination TR2-Fc Fusion Proteins

The 5′ portion of the TR2 containing the entire putative open readingframe of extracellular domain was amplified by polymerase chain reaction(Saiki, R. K. et al., Science 239:487 (1988)). For correctly orientedcloning, a HindIII site on the 5′ end of the forward primer and a Bg1IIsite on the 5′ end of the reverse primer were created. The Fc portion ofhuman IgG₁ was PCR-amplified from ARH-77 (ATCC™) cell RNA and cloned inSmaI site of pGem7 vector (Promega). The Fc fragment including hinge,CH₂ and CH₃ domain sequences contained a Bg1II site at its 5′ end and anXhoI site at its 3′ end. The HindIII-Bg1II fragment of TR2 cDNA wasinserted into the upstream of human IgG₁ Fc and an in frame fusion wasconfirmed by sequencing. The TR2-Fc fragment was released by digestingthe plasmid with HindIII-XhoI and cloned it into pcDNA3 expressionplasmid.

The TR2-Fc plasmid, linearized with PvuI, was transferred into NIH 3T3by the calcium phosphate co-precipitation method. After selection in 400μg/ml G418, neomycin-resistant colonies were picked and expanded. ELISAwith anti-human IgG₁ and Northern analysis with ³²P-labeled TR2 probewere used to select clones that produce high levels of TR2-Fc in thesupernatant. In some experiments, a slightly different engineered TR2-Fcproduced in Chinese hamster ovary (CHO) cells was used. The TR2-Fc waspurified by protein G chromatography, and the amino acid sequence ofN-terminus of the TR2-Fc fusion protein was determined by automaticpeptide sequencer (ABI). TR2-Fc was used to produce polyclonal rabbitanti-TR2 antibodies.

Blocking MLR-Mediated PBMC Proliferation

PBMC were isolated from three healthy adult volunteers by Ficollgradient centrifugation at 400×g for 30 minutes. PBMCs were recovered,washed in RPMI 1640 (GIBCO-BRL) supplemented with 10% FBS, 300 μg/mlL-glutamine and 50 μg/ml genetomycin, and adjusted to 1×10⁶ cells/ml fortwo donors and to 2×10⁵ cells/ml for the third donor.

Fifty μl of each cell suspension was added to 96-well (round bottom)plates (Falcon, Franklin Lakes, NS) together with 50 μl of TR2-Fc,IL-5R-Fc, anti-CD4 mAb or control mAb. Plates were incubated at 37 C in5% CO₂ for 96 hours. One μCi of [³H]-methylthymidine (ICN Biomedicals,Costa Mesa, Calif.) was then added for an additional 16 hours. Cellswere harvested and radioactivity was counted.

Results and Discussion

TR2 is a New Member of the TNFR Superfamily

FIG. 1A-1B (SEQ ID NO:2) shows the amino acid sequence of TR2 deducedfrom the longest open reading frame of one of the isolated cDNAs(HLHAB49). Comparison with other sequenced cDNAs and ESTs in thedatabase indicated potential allelic variants which resulted in aminoacid changes at positions 17 (either Arg or Lys) and 41 (either Ser orPhe) of the protein sequence shown in FIG. 1A-1B (amino acid residues−20 and 5 in SEQ ID NO:2).

The open reading frame encodes 283 amino acids with a calculatedmolecular weight of 30,417. The TR2 protein was expected to be areceptor. Therefore, the potential signal sequence and transmembranedomain were sought. A hydrophobic stretch of 23 amino acids towards theC terminus (amino acids 201-225) (FIG. 1A-1B) was assigned as atransmembrane domain because it made a potentially single helical span,but the signal sequence was less obvious. The potential ectodomain TR2was expressed in NIH 3T3 and CHO cells as a Fc-fusion protein, and theN-terminal amino acid sequence of the recombinant TR2-Fc protein wasdetermined in both cases. The N-terminal sequence of the processedmature TR2 started from amino acid 37, indicating that the first 36amino acids constituted the signal sequence (FIG. 1A-1B).

Using a polyclonal rabbit antibody raised to TR2, the molecular size ofnatural TR2 was determined to be 38 kD by Western analysis. Since theprotein backbone of processed TR2 would be composed of 247 amino acidswith an Mr of 26,000, the protein must be modified post-translationally.Two potential asparagine-linked glycosylation sites are located at aminoacid positions 110 and 173 (FIG. 1A-1B). Along with the other members ofthe TNFR family, TR2 contains the characteristic cysteine-rich motifswhich have been shown by X-ray crystallography (Banner et al., Cell73:431 (1993)) to represent a repetitive structural unit (Banner, D. W.et al., Cell 73:431 (1993)). FIG. 16 shows the potential TNFR domainaligned among TR2 (SEQ ID NO:2), TNFR-I (SEQ ID NO: 10), TNFR-II (SEQ IDNO: 11), CD40 (SEQ ID NO: 12) and 4-1BB (SEQ ID NO: 13). TR2 containedtwo perfect TNFR domain and two imperfect ones.

The TR2 cytoplasmic tail (TR2 cy) appears to be more closely related tothose of CD40cy and 4-1BBcy, and does not contain the death domain seenin the Fas and TNFR-I intracellular domains. Although the homology ismoderate, the Thr²⁶⁶ of TR2 is aligned with Thr²³³ of 4-1BB and Thr²⁵⁴of CD40. This may be significant because Inui et al., (Inui, S. et al.,Eur. J. Immunol. 20:1747 (1990)) found that Thr²⁵⁴ was essential forCD40 signal transduction and when the Thr²⁵⁴ of CD40 was mutated, theCD40 bd did not bind to the CD40cy (Hu, H. M. et al., J. Biol. Chem.269:30069 (1994)). Signals through 4-1BB and CD40 have been shown to becostimulatory to T cells and B cells respectively (Banchereau, J. andRousset, F., Nature 353:678 (1991); Hurtaldo, J. et al., J. Immunol.155:3360 (1995)).

TABLE 2 GENE EXPRESSION OF TR2 and OX40 IN TISSUES AND CELLS SOURCE GENETR2 OX-40 TISSUES (adult) Brain +/− − Heart + − Lung + − Thymus ++ −Spleen +++ − Liver + − Kidney + − Small Intestine +++ − Prostate ++ −Skeletal Muscle +/− Ovary + Pancreas + Colon + Thyroid + Spinal Cord +Trachea + Adrenal Gland + Lymph Node +++ − PRIMARY CELLS PBL, CD19+ ++ −PBL, CD8+ ++ − PBL, CD8+ (activated) ++ ++ PBL +++ PBL, CD4+ (activated)++ ++ Bone Marrow + − Monocyte ++ − Endothelial + − HEMATOPOIETIC CELLLINES Erythroid K562 − HEL + Myeloid KG1a (Promyeloblast) + + KG1(Myeloblast) ± ± PLB985 (Late myeloblast) − HL60 (Promyelocyte) ± − U937(Promonocyte) ± THP-1 (Monocyte) + − B-Lymphocyte REH (Pre-preB) ±−BJA-B (Early B, IgM) + Raji (Mature B, IgM) + IM-9 (Mature B, IgG₁) − −T-Lymphocyte Sup-T1 (CD4+) − Molt-3 (CD4+) ±− H9 (CD4+) + Jurkat(CD4+) + + no entry = not tested, − = not detected, ± to ++ = increasingamounts of RNA detectedTR2 RNA Expression

A human tissue RNA blot was used to determine tissue distribution of TR2RNA expression. TR2 RNA was detected in several tissues with arelatively high level in the lung, spleen and thymus (Table 2) but wasnot detected by this method in the brain, liver or skeletal muscle(Table 2). TR-2 was also expressed in monocytes, CD19⁺ B cells, andresting or PMA plus PHA-treated CD4⁺ or CD8⁺ T cells. It was only weaklyexpressed in bone marrow and endothelial cells (Tables 2 and 3),although expression was observed in the hematopoietic cell line KG1a(Table 2). For comparison, the tissue distribution of OX-40, anothermember of the TNFR superfamily, was examined (Table 2). Unlike TR2,OX-40 was not detected in any tissues examined, and was detected only inactivated T-cells and KG1a. Several cell lines were negative for TR2expression, including TF 274 (bone marrow stromal), MG 63 and TE 85(osteosarcoma), RL 95-2 (endometrial sarcoma), MCF-7 and T-47D (breastcancer cells), BE, HT 29 (colon cancer cells), HTB-11 and IMR-32(neuroblastoma), although TR2 was found in the rhabdosarcoma HTB-82(data not shown).

Several cell lines were examined for inducible TR2 expression. HL60,U937 and THP1, which belong to the myelomonocytic lineage, all increasedTR2 expression in response to the differentiation agents PMA or DMSO.Increases in expression in response to these agents were observed inKG1a and Jurkat cells. In contrast, PMA did not induce TR2 expression inMG63, but unexpectedly TNF-α did.

In almost all cases, the predominant mRNA was approximately 1.7 kb insize, although several higher molecular weight species could be detectedin some tissues. While many cDNAs and ESTs which were sequencedcontained insertions in the coding region indicative of partialsplicing, we only detected one major protein by Western blot, suggestingthat if these encode alternate proteins they are not evident in thecells we examined. The abundance of higher MW mRNAs raises thepossibility that TR2 may in part be regulated at the level of mRNAmaturation.

TABLE 3 RELATIVE ABUNDANCE (RA) of TR2 RNA IN VARIOUS TISSUE AND CELLTYPES Tissue or Cell Type RA Activated Macrophage (LPS) 22 Breast LymphNode 5 B Cell Lymphoma 5 Activated Monocytes 2 Activated T Cells 3Activated Neutrophil 2 Tonsils 5 Thymus 3 Anergic T-cell 1 Jurkat T-Cell3 Raji Cells (Cycloheximide Treated) 3 Atrophic Endometrium 1 BoneMarrow 1 Brain 1 Breast 1 CD34 Depleted Buffy Coat (Cord Blood) 1Cerebellum 1 Corpus Colosum 1 Caco-2 Cells (adenocarcinoma, colon) 1Fetal Dura Mater 1 Fetal Heart 1 Fetal Lung 2 Glioblastoma 1Hypothalamus, Schizophrenia 1 Infant Brain 2 Lung 2 Osteosarcoma 1Pancreas Tumor 1 Placenta 2 Small Intestine 1 Smooth Muscle 1 Stomach 2T-Cell Lymphoma 1 T-Cells 1 Testes 3 Testes Tumor 2 Tongue 1 UmbilicalVein Endothelial Cells 2 White Fat 3TR2 Maps at 1P36.2-P36.3

The FISH mapping procedure was applied to localize the TR2 gene to aspecific human chromosomal region. The assignment of a hybridizationsignal to the short arm of chromosome 1 was obtained with the aid ofDAPI banding. A total of 10 metatic figures. were photographed whichindicated that the TR2 gene is located on the chromosome 1 regionp36.2-p36.3. The TR2 position is in close proximity with CD30 (Smith, C.A. et al., Cell 73:1349-1360 (1993), 4-1BB (Kwon, B. S. et al., J.Immunol. 152:2256-2262 (1994); Goodwin, R. G. et al., Eur. J. Immunol.23:2631-2641 (1993), OX-40 (Birkeland, M. L. et al., Eur. J. Immunol.25:926-930 (1995), and TNFR-II (Baker, E. et al., Cytogenet. & CellGenet. 57:117-118 (1991), suggesting that it evolved through a localizedgene duplication event. Interestingly, all of these receptors havestimulatory phenotypes in T cells in response to cognate ligand binding,in contrast to Fas and TNFR-I which stimulate apoptosis. This promptedus to test if TR2 might be involved in lymphocyte stimulation.

TR2-Fc Interfaces with MLR-Mediated Proliferation of PBMC

To determine the possible involvement of cell surface TR2 with itsligand in lymphocyte proliferation, we examined allogeneic MLRproliferative responses. When TR-2-Fc was added to the culture, asignificant reduction of maximal responses was observed (p<0.05). Theaddition of TR2-Fc at 100 μg/ml inhibited the proliferation up to 53%.No significant inhibition of proliferation was observed with the controlIL-5R-Fc. Surprisingly, at high concentrations (10-100 μg/ml) IL-5R-Fcwas shown to enhance proliferation. An anti-CD4 mAb assayedsimultaneously inhibited MLR-mediated proliferation up to 60%, whereas acontrol anti-IL-5 mAb failed to inhibit the proliferation. It is wellknown that a major component of the MLR proliferative response is Tcell-dependent; hence, it would appear that inhibiting the interactionof TR2 with its ligand prevents optimal T lymphocyte activation andproliferation. The inhibition of MLR proliferation by TR2-Fc atconcentrations of 1-100 μg/ml compares favorably with biological effectsseen with other TNFR-Fc superfamily members such as CD40-Fc (unpublishedresults, Jeremy Harrop).

Hence, we have identified an additional member of the TNF receptorsuperfamily which either plays a direct role in T cell stimulation orbinds to a ligand which can stimulate T cell proliferation through oneor more receptors which may include TR2. Consistent with a direct rolefor TR2 is the similarity of the cytoplasmic domain with CD40 and 4-1BB.We are currently trying to identify this ligand to which TR2 binds inorder to clarify its role.

Example 7 Expression Pattern of TNF Receptor Expression in Human Tissue

Northern blot analysis is carried out to examine the levels ofexpression of TNF receptor in human tissues. Total cellular RNA samplesare isolated with RNAZOL™ B system (Biotecx Laboratories, Inc., Houston,Tex.). About 10 μg of total RNA isolated from each human tissuespecified is separated on 1% agarose gel and blotted onto a nylon filter(Sambrook, Fritsch, and Maniatis, Molecular Cloning, Cold Spring HarborPress, (1989)). The labeling reaction is done according to theStratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA ispurified with a Select-G 50 column (5 Prime-3 Prime, Inc., Boulder,Colo.). The filter is then hybridized with radioactive labeled fulllength TNF receptor gene at 1,000,000 cpm/ml in 0.5 M NaPO₄, pH 7.4 and7% SDS overnight at 65° C. After washing twice at room temperature andtwice at 60° C. with 0.5×SCC, 0.1% SDS, the filter is then exposed at−70° C. overnight with an intensifying screen.

Example 8 Cloning and Expression of the TNF Receptor and Extracellular(Soluble) TNF Soluble Form of the TNF Receptor Using the BaculovirusExpression System

The DNA sequence encoding the full length TNF receptor protein, ATCC™Accession No. 97059, and the soluble receptor nucleic acid are amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene (throughout this example TNF receptor is meant toinclude both the full-length receptor and the soluble form of thereceptor): The 5′ primer has the sequence 5′GCGCGGATCCACCATGGAGCCTCCTGGAGACTGG 3′ (SEQ ID NO:27) and contains aBamHI restriction enzyme site (in bold) followed by 3 nucleotidesresembling an efficient signal for the initiation of translation iseukaryotic cells (Kozak, M., J. Mol. Biol. 196:947-950 (1987) and whichis just behind the first 21 nucleotides of the TNF receptor gene (theinitiation codon for translation “ATG” is underlined.

The 3′ primer has the sequence 5′ GCGCGGTACCTCAGTGGGAGCTGCTGGTCCC 3′(SEQ ID NO:28) (for extracellular domain) and 5′GCGCGGTACCTCAGTGGTTTGGGCTCCTCCC 3′ (SEQ ID NO:29) (for full-lengthreceptor) and contains the cleavage site for the restrictionendonuclease Asp718 and 21 nucleotides complementary to the 3′non-translated sequence of the TNF receptor gene. The amplifiedsequences are isolated from a 1% agarose gel using a commerciallyavailable kit (“GENECLEAN™,” BIO 101 Inc., La Jolla, Calif.). Thefragments are then digested with the endonucleases BamHI and Asp718 andthen purified again on a 1% agarose gel. This fragment is designated F2.

The vector pRG1 (modification of pVL941 vector, discussed below) is usedfor the expression of the TNF receptor proteins using the baculovirusexpression system (for review see: Summers, M. D. and Smith, G. E. 1987,A manual of methods for baculovirus vectors and insect cell cultureprocedures, Texas Agricultural Experimental Station Bulletin No. 1555).This expression vector contains the strong polyhedron promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed bythe recognition sites for the restriction endonucleases BamHI andAsp718. The polyadenylation site of the simian virus (SV)40 is used forefficient polyadenylation. For an easy selection of recombinant virusesthe beta-galactosidase gene from E. coli is inserted in the sameorientation as the polyhedron promoter followed by the polyadenylationsignal of the polyhedron gene. The polyhedron sequences are flanked atboth sides by viral sequences for the cell-mediated homologousrecombination of cotransfected wild-type viral DNA. Many otherbaculovirus vectors could be used in place of pRG1 such as pAc373,pVL941 and pAcIM1 (Luckow, V. A. and Summers, M. D., Virology,170:31-39).

The plasmid is digested with the restriction enzymes BamHI and Asp718and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The DNA is then isolated from a 1% agarosegel using the commercially available kit (“GENECLEAN™,” BIO 101 Inc., LaJolla, Calif.). This vector DNA is designated V2.

Fragment F2 and the dephosphorylated plasmid V2 are ligated with T4 DNAligase. Sf9 cells are then transformed and cells identified thatcontained the plasmid (pBac TNF receptor) with the TNF receptor genesusing the enzymes BamHI and Asp718. The sequence of the cloned fragmentis confirmed by DNA sequencing.

5 μg of the plasmid pBac TNF receptor is cotransfected with 1.0 μg of acommercially available linearized baculovirus (“BACULOGOLD™ baculovirusDNA”, Pharmingen, San Diego, Calif.) using the lipofection method(Felgner, et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417 (1987)).

1 μg of BACULOGOLD™ virus DNA and 5 μg of the plasmid pBac TNF receptorsare mixed in a sterile well of a microtiter plate containing 50 μl ofserum free Grace's medium (Life Technologies Inc., Gaithersburg, Md.).Afterwards 10 μl LIPOFECTIN™ plus 90 μl Grace's medium are added, mixedand incubated for 15 minutes at room temperature. Then the transfectionmixture is added dropwise to the Sf9 insect cells (ATCC™ CRL 1711)seeded in a 35 mm tissue culture plate with 1 ml Grace's medium withoutserum. The plate is rocked back and forth to mix the newly addedsolution. The plate is then incubated for 5 hours at 27° C. After 5hours the transfection solution is removed from the plate and 1 ml ofGrace's insect medium supplemented with 10% fetal calf serum is added.The plate is put back into an incubator and cultivation continued at 27°C. for four days.

After four days the supernatant is collected and a plaque assayperformed similar as described by Summers and Smith (supra). As amodification an agarose gel with “BLUE GAL™” (Life Technologies Inc.,Gaithersburg, Md.) is used which allows an easy isolation of bluestained plaques. (A detailed description of “plaque assay” can also befound in the user's guide for insect cell culture and baculovirologydistributed by Life Technologies Inc., Gaithersburg, page 9-10).

Four days after the serial dilution, the viruses are added to the cellsand blue stained plaques are picked with the tip of an Eppendorfpipette. The agar containing the recombinant viruses are thenresuspended in an Eppendorf tube containing 200 μl of Grace's medium.The agar is removed by a brief centrifugation and the supernatantcontaining the recombinant baculoviruses is used to infect Sf9 cellsseeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then stored at 4° C.

Sf9 cells are grown in Grace's medium supplemented with 10%heat-inactivated FBS. The cells are infected with the recombinantbaculovirus V-TNF receptors at a multiplicity of infection (MOI) of 2.Six hours later the medium is removed and replaced with SF900 II mediumminus methionine and cysteine (Life Technologies Inc., Gaithersburg). 42hours later 5 μCi of ³⁵S-methionine and 5 μCi of ³⁵S cysteine (Amersham)are added. The cells are further incubated for 16 hours before they areharvested by centrifugation and the labeled proteins visualized bySDS-PAGE and autoradiography.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. A method for determining the level of a TR2 polypeptide in abiological sample from an individual comprising: (a) contacting thebiological sample from the individual with an antibody or fragmentthereof which binds to said polypeptide; (b) allowing a complex to formbetween said polypeptide and said antibody or fragment thereof; and (c)detecting said complex; wherein said TR2 polypeptide has an amino acidsequence selected from the group consisting of: (i) the amino acidsequence at positions from about −36 to about 247 in SEQ ID NO:2; (ii)the amino acid sequence at positions from about −35 to about 247 in SEQID NO:2; (iii) the amino acid sequence at positions from about 1 toabout 247 in SEQ ID NO:2; (iv) the amino acid sequence encoded by thecDNA clone contained in ATCC™ Deposit Number 97059; (v) the amino acidsequence of the mature TR2 polypeptide encoded by the cDNA clonecontained in ATCC™ Deposit Number 97059; (vi) the amino acid sequence atpositions from about 1 to about 164 in SEQ ID NO:2; (vii) the amino acidsequence at positions from about −36 to about 149 in SEQ ID NO:5; (viii)the amino acid sequence at positions from about −35 to about 149 in SEQID NO:5; (ix) the amino acid sequence at positions from about 1 to about149 in SEQ ID NO:5; (x) the amino acid sequence encoded by the cDNAclone contained in ATCC™ Deposit Number 97058; (xi) the amino acidsequence of the mature TR2 polypeptide encoded by the cDNA clonecontained in ATCC™ Deposit Number 97058; (xii) the amino acid sequencein SEQ ID NO:8; (xiii) the amino acid sequence at positions from about 2to about 136 in SEQ ID NO:8; and (xiv) the amino acid sequence encodedby the cDNA clone contained in ATCC™ Deposit Number
 97057. 2. The methodof claim 1, wherein said method is a radioimmunoassay.
 3. The method ofclaim 1, wherein said method is a competitive-binding assay.
 4. Themethod of claim 1, wherein said method is a Western blot.
 5. The methodof claim 1, wherein said method is an ELISA.
 6. The method of claim 1,wherein said antibody or fragment thereof is selected from the groupconsisting of: (a) a monoclonal antibody; (b) a polyclonal antibody; (c)a Fab fragment; and (d) a product of an Fab expression library.